bluuebunny/crossref_metadata_2025 huggingface dataset:
Cerebellar cortical inhibition and classical eyeblink conditioning
Authors: Bao Shaowen , Chen Lu , Kim Jeansok J. , Thompson Richard F.
Published: July 2002

The cerebellum is considered a brain structure in which memories for learned motor responses (e.g., conditioned eyeblink responses) are stored. Within the cerebellum, however, the relative importance of the cortex and the deep nuclei in motor learning/memory is not entirely clear. In this study, we show that the cerebellar cortex exerts both basal and stimulus-activated inhibition to the deep nuclei. Sequential application of a γ-aminobutyric acid type A receptor (GABA
A
R) agonist and a noncompetitive GABA
A
R antagonist allows selective blockade of stimulus-activated inhibition. By using the same sequential agonist and antagonist methods in behaving animals, we demonstrate that the conditioned response (CR) expression and timing are completely dissociable and involve different inhibitory inputs; although the basal inhibition modulates CR expression, the conditioned stimulus-activated inhibition is required for the proper timing of the CR. In addition, complete blockade of cerebellar deep nuclear GABA
A
Rs prevents CR acquisition. Together, these results suggest that different aspects of the memories for eyeblink CRs are encoded in the cerebellar cortex and the cerebellar deep nuclei.
Leptin-deficient (ob/ob) mice are protected from T cell-mediated hepatotoxicity: Role of tumor necrosis factor α and IL-18
Authors: Faggioni Raffaella , Jones-Carson Jessica , Reed David A. , Dinarello Charles A. , Feingold Kenneth R. , Grunfeld Carl , Fantuzzi Giamila
Published: July 2002

The role of leptin was investigated in two models of T cell-mediated hepatitis: the administration of Con A or ofPseudomonas aeruginosaexotoxin A (PEA). In both models, leptin-deficient (ob/ob) mice were protected from liver damage and showed lower induction of tumor necrosis factor (TNF) α and IL-18 compared with their lean littermates. Neutralization of TNF-α reduced induction of IL-18 by either Con A (70% reduction) or PEA (40% reduction). Pretreatment of lean mice with either soluble TNF receptors or with an anti-IL-18 antiserum significantly reduced Con A- and PEA-induced liver damage. The simultaneous neutralization of TNF-α and IL-18 fully protected the mice against liver toxicity. However, neutralization of either IL-18 or TNF-α did not inhibit Con A-induced production of IFN-γ. Thymus atrophy and alterations in the number of circulating lymphocytes and monocytes were observed inob/obmice. Exogenous leptin replacement restored the responsiveness ofob/obmice to Con A and normalized their lymphocyte and monocyte populations. These results demonstrate that leptin deficiency leads to reduced production of TNF-α and IL-18 associated with reduced T cell-mediated hepatotoxicity. In addition, both TNF-α and IL-18 appear to be essential mediators of T cell-mediated liver injury.
Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification
Authors: Zhong Xiao-bo , Lizardi Paul M. , Huang Xiao-hua , Bray-Ward Patricia L. , Ward David C.
Published: July 2002

Rolling circle amplification (RCA) is a surface-anchored DNA
replication reaction that can be exploited to visualize single
molecular recognition events. Here we report the use of RCA to
visualize target DNA sequences as small as 50 nts in peripheral blood
lymphocytes or in stretched DNA fibers. Three unique target sequences
within the cystic fibrosis transmembrane conductance regulator gene
could be detected simultaneously in interphase nuclei, and could be
ordered in a linear map in stretched DNA. Allele-discriminating
oligonucleotide probes in conjunction with RCA also were used to
discriminate wild-type and mutant alleles in the cystic fibrosis
transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome
genes in the nuclei of cultured cells or in DNA fibers. These
observations demonstrate that signal amplification by RCA can be
coupled to nucleic acid hybridization and multicolor fluorescence
imaging to detect single nucleotide changes in DNA within a cytological
context or in single DNA molecules. This provides a means for direct
physical haplotyping and the analysis of somatic mutations on a
cell-by-cell basis.
Gangliosides are functional nerve cell ligands for myelin-associated glycoprotein (MAG), an inhibitor of nerve regeneration
Authors: Vyas Alka A. , Patel Himatkumar V. , Fromholt Susan E. , Heffer-Lauc Marija , Vyas Kavita A. , Dang Jiyoung , Schachner Melitta , Schnaar Ronald L.
Published: July 2002

Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (
i
) neuraminidase treatment of the neurons; (
ii
) blocking neuronal ganglioside biosynthesis; (
iii
) genetically modifying the terminal structures of nerve cell surface gangliosides; and (
iv
) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering.
Development of an extrachromosomal cloning vector system for use in
Borrelia burgdorferi
Authors: Sartakova Marina , Dobrikova Elena , Cabello Felipe C.
Published: July 2002

Molecular genetic analysis of
Borrelia burgdorferi
, the cause of Lyme disease, has been hampered by the absence of any means of efficient generation, identification, and complementation of chromosomal and plasmid null gene mutants. The similarity of borrelial G + C content to that of Gram-positive organisms suggested that a wide-host-range plasmid active in Gram-positive bacteria might also be recognized by borrelial DNA replication machinery. One such plasmid, pGK12, is able to propagate in both Gram-positive and Gram-negative bacteria and carries erythromycin and chloramphenicol resistance markers. pGK12 propagated extrachromosomally in
B. burgdorferi
B31 after electroporation but conferred only erythromycin resistance. pGK12 was used to express enhanced green fluorescent protein in B31 under the control of the
flaB
promoter.
Escherichia coli
transformed with pGK12 DNA extracted from B31 expressing only erythromycin resistance developed both erythromycin and chloramphenicol resistance, and plasmid DNA isolated from these transformed
E. coli
had a restriction pattern similar to the original pGK12. Our data indicate that the replicons of pGK12 can provide the basis to continue developing efficient genetic systems for
B. burgdorferi
together with the erythromycin resistance and reporter
egfp
genes.
Cancer-predisposing mutations within the RING domain of BRCA1: Loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity
Authors: Ruffner Heinz , Joazeiro Claudio A. P. , Hemmati Dan , Hunter Tony , Verma Inder M.
Published: July 2002

BRCA1 is a breast and ovarian cancer-specific tumor suppressor that
seems to be involved in transcription and DNA repair. Here we report
that BRCA1 exhibits a bona fide ubiquitin (Ub) protein ligase (E3)
activity, and that cancer-predisposing mutations within the BRCA1 RING
domain abolish its Ub ligase activity. Furthermore, these mutants are
unable to reverse γ-radiation hypersensitivity of BRCA1-null human
breast cancer cells, HCC1937. Additionally, these mutations within the
BRCA1 RING domain are not capable of restoring a G
2
+ M
checkpoint in HCC1937 cells. These results establish a link between Ub
protein ligase activity and γ-radiation protection function of BRCA1,
and provide an explanation for why mutations within the BRCA1 RING
domain predispose to cancer. Furthermore, we propose that the analysis
of the Ub ligase activity of RING-domain mutations identified in
patients may constitute an assay to predict predisposition to cancer.
Inverse radiation dose-rate effects on somatic and germ-line mutations and DNA damage rates
Authors: Vilenchik Michael M. , Knudson Alfred G.
Published: July 2002

The mutagenic effect of low linear energy transfer ionizing radiation is reduced for a given dose as the dose rate (DR) is reduced to a low level, a phenomenon known as the direct DR effect. Our reanalysis of published data shows that for both somatic and germ-line mutations there is an opposite, inverse DR effect, with reduction from low to very low DR, the overall dependence of induced mutations being parabolically related to DR, with a minimum in the range of 0.1 to 1.0 cGy/min (rule 1). This general pattern can be attributed to an optimal induction of error-free DNA repair in a DR region of minimal mutability (MMDR region). The diminished activation of repair at very low DRs may reflect a low ratio of induced (“signal”) to spontaneous background DNA damage (“noise”). Because two common DNA lesions, 8-oxoguanine and thymine glycol, were already known to activate repair in irradiated mammalian cells, we estimated how their rates of production are altered upon radiation exposure in the MMDR region. For these and other abundant lesions (abasic sites and single-strand breaks), the DNA damage rate increment in the MMDR region is in the range of 10% to 100% (rule 2). These estimates suggest a genetically programmed optimatization of response to radiation in the MMDR region.
Tomato
Ve
disease resistance genes encode cell surface-like receptors
Authors: Kawchuk Lawrence M. , Hachey John , Lynch Dermot R. , Kulcsar Frank , van Rooijen Gijs , Waterer Doug R. , Robertson Albert , Kokko Eric , Byers Robert , Howard Ronald J. , Fischer Rainer , Prüfer Dirk
Published: July 2002

In tomato,
Ve
is implicated in race-specific
resistance to infection by
Verticillium
species causing
crop disease. Characterization of the
Ve
locus involved
positional cloning and isolation of two closely linked inverted genes.
Expression of individual
Ve
genes in susceptible potato
plants conferred resistance to an aggressive race 1 isolate of
Verticillium albo-atrum
. The deduced primary structure
of
Ve
1 and
Ve
2 included a hydrophobic
N-terminal signal peptide, leucine-rich repeats containing 28 or 35
potential glycosylation sites, a hydrophobic membrane-spanning domain,
and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ
endocytosis signals (φ is an amino acid with a hydrophobic side
chain). A leucine zipper-like sequence occurs in the hydrophobic
N-terminal signal peptide of
Ve
1 and a Pro-Glu-Ser-Thr
(PEST)-like sequence resides in the C-terminal domain of
Ve
2. These structures suggest that the
Ve
genes encode a class of cell-surface glycoproteins with
receptor-mediated endocytosis-like signals and leucine zipper or PEST
sequences.
ICOS-ligand, expressed on human endothelial cells, costimulates Th1 and Th2 cytokine secretion by memory CD4+T cells
Authors: Khayyamian Saman , Hutloff Andreas , Büchner Kerstin , Gräfe Michael , Henn Volker , Kroczek Richard A. , Mages Hans W.
Published: July 2002

Endothelial cells (EC) play a central role in inflammatory immune responses and efficiently induce effector functions in T cells, despite lacking the classical costimulatory ligands CD80 and CD86. By using the mAb HIL-131 we now demonstrate that human inducible costimulator-ligand (ICOS-L), a molecule related to CD80/CD86, is constitutively expressed on human ECin vivo.In vitro, ICOS-L expression was strongly enhanced on human umbilical vein EC and microvascular EC by the inflammatory cytokines tumor necrosis factor α and IL-1β, and to a lower extent by stimulation of EC by CD40 or lipopolysaccharide. Coculture of MHC class II+EC with resting memory CD4+T cells in the presence of superantigen led to a marked up-regulation of ICOS on T cells and to the production of Th1 (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10, IL-13). When these cocultures were performed in the presence of the inhibitory mAb HIL-131, secretion of all cytokines was reduced by about 50–80%, indicating that ICOS-L is a major costimulator in EC-mediated T cell activation. Taken together, our data suggest an important physiological role of ICOS-L in the reactivation of effector/memory T cells on the endothelium controlling the entry of immune cells into inflamed tissue.
Jewish and Middle Eastern non-Jewish populations share a common pool of Y-chromosome biallelic haplotypes
Authors: Hammer M. F. , Redd A. J. , Wood E. T. , Bonner M. R. , Jarjanazi H. , Karafet T. , Santachiara-Benerecetti S. , Oppenheim A. , Jobling M. A. , Jenkins T. , Ostrer H. , Bonné-Tamir B.
Published: July 2002

Haplotypes constructed from Y-chromosome markers were used to trace the paternal origins of the Jewish Diaspora. A set of 18 biallelic polymorphisms was genotyped in 1,371 males from 29 populations, including 7 Jewish (Ashkenazi, Roman, North African, Kurdish, Near Eastern, Yemenite, and Ethiopian) and 16 non-Jewish groups from similar geographic locations. The Jewish populations were characterized by a diverse set of 13 haplotypes that were also present in non-Jewish populations from Africa, Asia, and Europe. A series of analyses was performed to address whether modern Jewish Y-chromosome diversity derives mainly from a common Middle Eastern source population or from admixture with neighboring non-Jewish populations during and after the Diaspora. Despite their long-term residence in different countries and isolation from one another, most Jewish populations were not significantly different from one another at the genetic level. Admixture estimates suggested low levels of European Y-chromosome gene flow into Ashkenazi and Roman Jewish communities. A multidimensional scaling plot placed six of the seven Jewish populations in a relatively tight cluster that was interspersed with Middle Eastern non-Jewish populations, including Palestinians and Syrians. Pairwise differentiation tests further indicated that these Jewish and Middle Eastern non-Jewish populations were not statistically different. The results support the hypothesis that the paternal gene pools of Jewish communities from Europe, North Africa, and the Middle East descended from a common Middle Eastern ancestral population, and suggest that most Jewish communities have remained relatively isolated from neighboring non-Jewish communities during and after the Diaspora.
Myogenic stem cell function is impaired in mice lacking the
forkhead
/winged helix protein MNF
Authors: Garry Daniel J. , Meeson Annette , Elterman Joel , Zhao Yuhe , Yang Peng , Bassel-Duby Rhonda , Williams R. Sanders
Published: July 2002

Myocyte nuclear factor (MNF) is a winged helix transcription factor that is expressed selectively in myogenic stem cells (satellite cells) of adult animals. Using a gene knockout strategy to generate a functional null allele at the
Mnf
locus, we observed that mice lacking MNF are viable, but severely runted. Skeletal muscles of
Mnf
−/− animals are atrophic, and satellite cell function is impaired. Muscle regeneration after injury is delayed and incomplete, and the normal timing of expression of cell cycle regulators and myogenic determination genes is dysregulated.
Mnf
mutant mice were intercrossed with
mdx
mice that lack dystrophin and exhibit only a subtle myopathic phenotype. In contrast,
mdx
mice that also lack MNF die in the first few weeks of life with a severe myopathy. Haploinsufficiency at the
Mnf
locus (
Mnf
+/−) also exacerbates the
mdx
phenotype to more closely resemble Duchenne's muscular dystrophy in humans. We conclude that MNF acts to regulate genes that coordinate the proliferation and differentiation of myogenic stem cells after muscle injury. Animals deficient in MNF may prove useful for evaluation of potential therapeutic interventions to promote muscle regeneration for patients having Duchenne's muscular dystrophy.
Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia
Authors: Shimada Takashi , Mizutani Satoru , Muto Takanori , Yoneya Takashi , Hino Rieko , Takeda Shu , Takeuchi Yasuhiro , Fujita Toshiro , Fukumoto Seiji , Yamashita Takeyoshi
Published: July 2002

Tumor-induced osteomalacia (TIO) is one of the paraneoplastic
diseases characterized by hypophosphatemia caused by renal phosphate
wasting. Because removal of responsible tumors normalizes phosphate
metabolism, an unidentified humoral phosphaturic factor is believed to
be responsible for this syndrome. To identify the causative factor of
TIO, we obtained cDNA clones that were abundantly expressed only in a
tumor causing TIO and constructed tumor-specific cDNA contigs. Based on
the sequence of one major contig, we cloned 2,270-bp cDNA, which turned
out to encode fibroblast growth factor 23 (FGF23). Administration of
recombinant FGF23 decreased serum phosphate in mice within 12 h.
When Chinese hamster ovary cells stably expressing FGF23 were s.c.
implanted into nude mice, hypophosphatemia with increased renal
phosphate clearance was observed. In addition, a high level of serum
alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone,
and impairment of body weight gain became evident. Histological
examination showed marked increase of osteoid and widening of growth
plate. Thus, continuous production of FGF23 reproduced clinical,
biochemical, and histological features of TIO
in vivo
.
Analyses for recombinant FGF23 products produced by Chinese hamster
ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif.
Recent genetic study indicates that missense mutations in this RXXR
motif of FGF23 are responsible for autosomal dominant hypophosphatemic
rickets, another hypophosphatemic disease with similar features to TIO.
We conclude that overproduction of FGF23 causes TIO, whereas mutations
in the FGF23 gene result in autosomal dominant hypophosphatemic rickets
possibly by preventing proteolytic cleavage and enhancing biological
activity of FGF23.
The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations
Authors: Ferguson David O. , Sekiguchi JoAnn M. , Chang Sandy , Frank Karen M. , Gao Yijie , DePinho Ronald A. , Alt Frederick W.
Published: July 2002

We have used spectral karyotyping to assess potential roles of three different components of the nonhomologous DNA end-joining pathway in the maintenance of genomic stability in mouse embryonic fibroblasts (MEFs). MEFs homozygous for mutations that inactivate either DNA ligase IV (Lig4) or Ku70 display dramatic genomic instability, even in the absence of exogenous DNA damaging agents. These aberrant events range from chromosomal fragmentation to nonreciprocal translocations that can involve several chromosomes. DNA-dependent protein kinase catalytic subunit deficiency also promotes genome instability. Deficiency for the p53 cell cycle checkpoint protein has little effect on spontaneous levels of chromosomal instability in Lig4-deficient fibroblasts. However, in the context of ionizing radiation treatment, p53 deficiency allowed visualization of massive acute chromosomal destruction in Lig4-deficient MEFs, which in surviving cells manifested as frequent nonreciprocal translocations. We conclude that nonhomologous DNA end-joining plays a crucial role as a caretaker of the mammalian genome, and that an alternative repair pathway exists that often leads to nonreciprocal translocations.
CtBP-dependent activities of the short-range Giant repressor in the
Drosophila
embryo
Authors: Nibu Yutaka , Levine Michael S.
Published: July 2002

There are at least three short-range gap repressors in the
precellular
Drosophila
embryo: Krüppel, Knirps,
and Giant. Krüppel and Knirps contain related repression motifs,
PxDLSxH and PxDLSxK, respectively, which mediate interactions with the
dCtBP corepressor protein. Here, we present evidence that Giant might
also interact with dCtBP. The misexpression of Giant in ventral regions
of transgenic embryos results in the selective repression of
eve
stripe 5. A
stripe5-lacZ
transgene
exhibits an abnormal staining pattern in
dCtBP
mutants
that is consistent with attenuated repression by Giant. The analysis of
Gal4-Giant fusion proteins identified a minimal repression domain that
contains a sequence motif, VLDLS, which is conserved in at least two
other sequence-specific repressors. Removal of this sequence from the
native Giant protein does not impair its repression activity in
transgenic embryos. We propose that Giant-dCtBP interactions might be
indirect and mediated by an unknown bZIP subunit that forms a
heteromeric complex with Giant. We also suggest that the VLDLS motif
recruits an as yet unidentified corepressor protein.
Chromosome translocations and covert leukemic clones are generated during normal fetal development
Authors: Mori Hiroshi , Colman Susan M. , Xiao Zhijian , Ford Anthony M. , Healy Lyn E. , Donaldson Craig , Hows Jill M. , Navarrete Cristina , Greaves Mel
Published: July 2002

Studies on monozygotic twins with concordant leukemia and retrospective scrutiny of neonatal blood spots of patients with leukemia indicate that chromosomal translocations characteristic of pediatric leukemia often arise prenatally, probably as initiating events. The modest concordance rate for leukemia in identical twins (≈5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and/or genetic events are required for clinically overt leukemia development. This notion leads to the prediction that chromosome translocations, functional fusion genes, and preleukemic clones should be present in the blood of healthy newborns at a rate that is significantly greater than the cumulative risk of the corresponding leukemia. Using parallel reverse transcriptase–PCR and real-time PCR (Taqman) screening, we find that the common leukemia fusion genes,
TEL-AML1
or
AML1-ETO
, are present in cord bloods at a frequency that is 100-fold greater than the risk of the corresponding leukemia. Single-cell analysis by cell enrichment and immunophenotype/fluorescence
in situ
hybridization multicolor staining confirmed the presence of translocations in restricted cell types corresponding to the B lymphoid or myeloid lineage of the leukemias that normally harbor these fusion genes. The frequency of positive cells (10
−4
to 10
−3
) indicates substantial clonal expansion of a progenitor population. These data have significant implications for the pathogenesis, natural history, and etiology of childhood leukemia.
Down-regulation of p21
WAF1/CIP1
or p27
Kip1
abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells
Authors: Cariou Sandrine , Donovan Jeffrey C. H. , Flanagan W. M. , Milic Andrea , Bhattacharya Nandita , Slingerland Joyce M.
Published: July 2002

Estrogens and antiestrogens influence the G
1
phase of the cell cycle. In MCF-7 breast cancer cells, estrogen stimulated cell cycle progression through loss of the kinase inhibitor proteins (KIPs) p27 and p21 and through G
1
cyclin-dependent kinase (cdk) activation. Treatment with antiestrogen drugs, Tamoxifen or ICI 182780, caused cell cycle arrest, with up-regulation of both p21 and p27 levels, an increase in their binding to cyclin E–cdk2, and kinase inhibition. The requirement for these KIPs in the arrests induced by estradiol depletion or by antiestrogens was investigated with antisense. Antisense inhibition of p21 or p27 expression in estradiol-depleted or antiestrogenarrested MCF-7 led to abrogation of cell cycle arrest, with loss of cyclin E-associated KIPs, activation of cyclin E–cdk2, and S phase entrance. These data demonstrate that depletion of either p21 or p27 can mimic estrogen-stimulated cell cycle activation and indicate that both of these KIPs are critical mediators of the therapeutic effects of antiestrogens in breast cancer.
A hebbian form of long-term potentiation dependent on mGluR1a in hippocampal inhibitory interneurons
Authors: Perez Yaël , Morin France , Lacaille Jean-Claude
Published: July 2002

Hippocampal inhibitory interneurons play important roles in controlling the excitability and synchronization of pyramidal cells, but whether they express long-term synaptic plasticity that contributes to hippocampal network function remains uncertain. We found that pairing postsynaptic depolarization with θ-burst stimulation induced long-term potentiation (LTP) of putative single-fiber excitatory postsynaptic currents in interneurons. Either postsynaptic depolarization or θ-burst stimulation alone failed to induce LTP. LTP was expressed as a decrease in failure rates and an increase in excitatory postsynaptic current amplitude, independent of
N
-methyl-
d
-aspartate receptors, and dependent on metabotropic glutamate receptors subtype 1a. LTP was induced specifically in interneurons in stratum oriens and not in interneurons of stratum radiatum/lacunosum-moleculare. Thus, excitatory synapses onto specific subtypes of inhibitory interneurons express a new form of hebbian LTP that will contribute to hippocampal network plasticity.
Failed retrograde transport of NGF in a mouse model of Down's syndrome: Reversal of cholinergic neurodegenerative phenotypes following NGF infusion
Authors: Cooper Jonathan D. , Salehi Ahmad , Delcroix Jean-Dominique , Howe Charles L. , Belichenko Pavel V. , Chua-Couzens Jane , Kilbridge Joshua F. , Carlson Elaine J. , Epstein Charles J. , Mobley William C.
Published: July 2002

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive decline in Alzheimer's disease and Down's syndrome. With aging, the partial trisomy 16 (Ts65Dn) mouse model of Down's syndrome exhibited reductions in BFCN size and number and regressive changes in the hippocampal terminal fields of these neurons with respect to diploid controls. The changes were associated with significantly impaired retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain. Intracerebroventricular NGF infusion reversed well established abnormalities in BFCN size and number and restored the deficit in cholinergic innervation. The findings are evidence that even BFCNs chronically deprived of endogenous NGF respond to an intervention that compensates for defective retrograde transport. We suggest that age-related cholinergic neurodegeneration may be a treatable disorder of failed retrograde NGF signaling.
Investigating stem cells in human colon by using methylation patterns
Authors: Yatabe Yasushi , Tavaré Simon , Shibata Darryl
Published: July 2002

The stem cells that maintain human colon crypts are poorly characterized. To better determine stem cell numbers and how they divide, epigenetic patterns were used as cell fate markers. Methylation exhibits somatic inheritance and random changes that potentially record lifelong stem cell division histories as binary strings or tags in adjacent CpG sites. Methylation tag contents of individual crypts were sampled with bisulfite sequencing at three presumably neutral loci. Methylation increased with aging but varied between crypts and was mosaic within single crypts. Some crypts appeared to be quasi-clonal as they contained more unique tags than expected if crypts were maintained by single immortal stem cells. The complex epigenetic patterns were more consistent with a crypt niche model wherein multiple stem cells were present and replaced through periodic symmetric divisions. Methylation tags provide evidence that normal human crypts are long-lived, accumulate random methylation errors, and contain multiple stem cells that go through “bottlenecks” during life.
Discovering functional relationships between RNA expression and chemotherapeutic susceptibility using relevance networks
Authors: Butte Atul J. , Tamayo Pablo , Slonim Donna , Golub Todd R. , Kohane Isaac S.
Published: July 2002

In an effort to find gene regulatory networks and clusters of genes
that affect cancer susceptibility to anticancer agents, we joined a
database with baseline expression levels of 7,245 genes measured by
using microarrays in 60 cancer cell lines, to a database with the
amounts of 5,084 anticancer agents needed to inhibit growth of those
same cell lines. Comprehensive pair-wise correlations were calculated
between gene expression and measures of agent susceptibility.
Associations weaker than a threshold strength were removed, leaving
networks of highly correlated genes and agents called relevance
networks. Hypotheses for potential single-gene determinants of
anticancer agent susceptibility were constructed. The effect of random
chance in the large number of calculations performed was empirically
determined by repeated random permutation testing; only associations
stronger than those seen in multiply permuted data were used in
clustering. We discuss the advantages of this methodology over
alternative approaches, such as phylogenetic-type tree clustering and
self-organizing maps.
A
Saccharomyces
gene family involved in invasive growth, cell–cell adhesion, and mating
Authors: Guo Bing , Styles Cora A. , Feng Qinghua , Fink Gerald R.
Published: July 2002

The cell wall of bakers' yeast contains a family of
glycosyl-phosphatidylinositol (GPI)-linked glycoproteins of domain
structure similar to the adhesins of pathogenic fungi. In wild-type
cells each of these proteins has a unique function in different
developmental processes (mating, invasive growth, cell–cell adhesion,
or filamentation). What unifies these developmental events is adhesion,
either to an inert substrate or to a cell. Although they differ in
their specificities, many of these proteins can substitute for each
other when overexpressed. For example, Flo11p is required during
vegetative growth for haploid invasion and diploid filamentation,
whereas Fig2p is required for mating. When overexpressed, Flo11p and
Fig2p can function in mating, invasion, filamentation, and
flocculation. The ability of Flo11p to supply Fig2p function in mating
depends on its intracellular localization to the mating projection,
where Fig2p normally functions in the adhesion of mating cells. Our
data show that even distant family members retain the ability to carry
out disparate functions if localized and expressed appropriately.
Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs
Authors: Ochsenbein Adrian F. , Pinschewer Daniel D. , Sierro Sophie , Horvath Edit , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

Memory is a hallmark of immunity. Memory carried by antibodies is largely responsible for protection against reinfection with most known acutely lethal infectious agents and is the basis for most clinically successful vaccines. However, the nature of long-term B cell and antibody memory is still unclear. B cell memory was studied here after infection of mice with the rabies-like cytopathic vesicular stomatitis virus, the noncytopathic lymphocytic choriomeningitis virus (Armstrong and WE), and after immunization with various inert viral antigens inducing naive B cells to differentiate either to plasma cells or memory B cells in germinal centers of secondary lymphoid organs. The results show that in contrast to very low background levels against internal viral antigens, no significant neutralizing antibody memory was observed in the absence of antigen and suggest that memory B cells (i) are long-lived in the absence of antigen, nondividing, and relatively resistant to irradiation, and (ii) must be stimulated by antigen to differentiate to short-lived antibody-secreting plasma cells, a process that is also efficient in the bone marrow and always depends on radiosensitive, specific T help. Therefore, for vaccines to induce long-term protective antibody titers, they need to repeatedly provide, or continuously maintain, antigen in minimal quantities over a prolonged time period in secondary lymphoid organs or the bone marrow for sufficient numbers of long-lived memory B cells to mature to short-lived plasma cells.
FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction
Authors: Desai Bimal N. , Myers Benjamin R. , Schreiber Stuart L.
Published: July 2002

FKBP12-rapamycin associated protein (FRAP, also known as mTOR or RAFT) is the founding member of the phosphatidylinositol kinase-related kinase family and functions as a sensor of physiological signals that regulate cell growth. Signals integrated by FRAP include nutrients, cAMP levels, and osmotic stress, and cellular processes affected by FRAP include transcription, translation, and autophagy. The mechanisms underlying the integration of such diverse signals by FRAP are largely unknown. Recently, FRAP has been reported to be regulated by mitochondrial dysfunction and depletion of ATP levels. Here we show that exposure of cells to hyperosmotic conditions (and to glucose-deficient growth medium) results in rapid and reversible dissipation of the mitochondrial proton gradient. These results suggest that the ability of FRAP to mediate osmotic stress response (and glucose deprivation response) is by means of an intermediate mitochondrial dysfunction. We also show that in addition to cytosolic FRAP a large portion of FRAP associates with the mitochondrial outer membrane. The results support the existence of a stress-sensing module consisting of mitochondria and mitochondrial outer membrane-associated FRAP. This module allows the cell to integrate a variety of stress signals that affect mitochondrial function and regulate a growth checkpoint involving p70 S6 kinase.
Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons.
Authors: Kelly J F , Furukawa K , Barger S W , Rengen M R , Mark R J , Blanc E M , Roth G S , Mattson M P
Published: July 2002

Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C, resulting in the liberation of inositol trisphosphate and Ca2+ release from intracellular stores. We now report that amyloid beta-peptide (Abeta), which forms plaques in the brain in AD, impairs muscarinic receptor activation of G proteins in cultured rat cortical neurons. Exposure of rodent fetal cortical neurons to Abeta25-35 and Abeta1-40 resulted in a concentration and time-dependent attenuation of carbachol-induced GTPase activity without affecting muscarinic receptor ligand binding parameters. Downstream events in the signal transduction cascade were similarly attenuated by Abeta. Carbachol-induced accumulation of inositol phosphates (IP, IP2, IP3, and IP4) was decreased and calcium imaging studies revealed that carbachol-induced release of calcium was severely impaired in neurons pretreated with Abeta. Muscarinic cholinergic signal transduction was disrupted with subtoxic levels of exposure to AP. The effects of Abeta on carbachol-induced GTPase activity and calcium release were attenuated by antioxidants, implicating free radicals in the mechanism whereby Abeta induced uncoupling of muscarinic receptors. These data demonstrate that Abeta disrupts muscarinic receptor coupling to G proteins that mediate induction of phosphoinositide accumulation and calcium release, findings that implicate Abeta in the impairment of cholinergic transmission that occurs in AD.
Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.
Authors: Mrksich M , Chen C S , Xia Y , Dike L E , Ingber D E , Whitesides G M
Published: July 2002

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.
Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells.
Authors: Matayoshi A , Brown C , DiPersio J F , Haug J , Abu-Amer Y , Liapis H , Kuestner R , Pacifici R
Published: July 2002

Osteoclastogenesis is a complex process that is facilitated by bone marrow stromal cells (SCs). To determine if SCs are an absolute requirement for the differentiation of human hematopoietic precursors into fully mature, osteoclasts (OCs), CD34+ cells were mobilized into the peripheral circulation with granulocyte colony-stimulating factor, harvested by leukapheresis, and purified by magnetic-activated cell sorting. This procedure yields a population of CD34+ cells that does not contain SC precursors, as assessed by the lack of expression of the SC antigen Stro-1, and that differentiates only into hematopoietic cells. We found that CD34+, Stro-1- cells cultured with a combination of granulocyte/macrophage colony-stimulating factor, interleukin 1, and interleukin 3 generated cells that fulfill current criteria for the characterization of OCs, including multinucleation, presence of tartrate-resistant acid phosphatase, and expression of the calcitonin and vitronectin receptors and of pp60c-src tyrosine kinase. These OCs also expressed mRNA for the noninserted isoform of the calcitonin receptor and excavated characteristic resorption pits in devitalized bone slices. These data demonstrate that accessory SCs are not essential for human osteoclastogenesis and that granulocyte colony-stimulating factor treatment mobilizes OC precursors into the peripheral circulation.
A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases.
Authors: Yang S W , Burgin A B , Huizenga B N , Robertson C A , Yao K C , Nash H A
Published: July 2002

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.
Cross-talk between the insulin and angiotensin signaling systems.
Authors: Velloso L A , Folli F , Sun X J , White M F , Saad M J , Kahn C R
Published: July 2002

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator of cardiac, vascular, and renal function. Following injection of AII into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the AT1 receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and p110 subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects of AII are inhibited by AT1 receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.
In vitro
activation of Stat3 by epidermal growth factor receptor kinase
Authors: Park Ohkmae K. , Schaefer Timothy S. , Nathans Daniel
Published: July 2002

Stat proteins are SH2 domain-containing transcription factors that
are activated in cells by various cytokines and growth factors. In the
case of cytokines whose receptors lack protein kinase activity,
phosphorylation–activation is mediated by members of the JAK family of
tyrosine protein kinases. In the case of growth factors whose receptors
have intrinsic tyrosine protein kinase activity, it is thought that
Stat proteins can be activated either directly by the receptor or
indirectly through JAK proteins. To test the possibility of direct
activation, we have used purified Stat3α, Stat3β, and epidermal
growth factor receptor kinase produced in recombinant
baculovirus-infected Sf9 insect cells. The Stat proteins formed a
stable complex with the receptor kinase, and they were phosphorylated
on tyrosine by the receptor kinase and activated for binding to DNA,
properties shared with Stat proteins purified from Sf9 cells
coexpressing JAK1 or JAK2. Both JAK-phosphorylated Stat3β and
Stat3β phosphorylated
in vitro
by the receptor kinase
were 20–50 times more active on a molar basis for DNA binding than
phosphorylated Stat3α. We conclude that Stat3 isoforms can be
directly phosphorylated and thereby activated
in vitro
by the epidermal growth factor receptor kinase.
Numerical representations in primates.
Authors: Hauser M D , MacNeilage P , Ware M
Published: July 2002

Research has demonstrated that human infants and nonhuman primates have a rudimentary numerical system that enables them to count objects or events. More recently, however, studies using a preferential looking paradigm have suggested that preverbal human infants are capable of simple arithmetical operations, such as adding and subtracting a small number of visually presented objects. These findings implicate a relatively sophisticated representational system in the absence of language. To explore the evolutionary origins of this capacity, we present data from an experiment with wild rhesus monkeys (Macaca mulatta) that methodologically mirrors those conducted on human infants. Results suggest that rhesus monkeys detect additive and subtractive changes in the number of objects present in their visual field. Given the methodological and empirical similarities, it appears that nonhuman primates such as rhesus monkeys may also have access to arithmetical representations, although alternative explanations must be considered for both primate species.
Cloning, expression, and properties of the microtubule-stabilizing protein STOP.
Authors: Bosc C , Cronk J D , Pirollet F , Watterson D M , Haiech J , Job D , Margolis R L
Published: July 2002

Nerve cells contain abundant subpopulations of cold-stable microtubules. We have previously isolated a calmodulin-regulated brain protein, STOP (stable tubule-only polypeptide), which reconstitutes microtubule cold stability when added to cold-labile microtubules in vitro. We have now cloned cDNA encoding STOP. We find that STOP is a 100.5-kDa protein with no homology to known proteins. The primary structure of STOP includes two distinct domains of repeated motifs. The central region of STOP contains 5 tandem repeats of 46 amino acids, 4 with 98% homology to the consensus sequence. The STOP C terminus contains 28 imperfect repeats of an 11-amino acid motif. STOP also contains a putative SH3-binding motif close to its N terminus. In vitro translated STOP binds to both microtubules and Ca2+-calmodulin. When STOP cDNA is expressed in cells that lack cold-stable microtubules, STOP associates with microtubules at 37 degrees C, and stabilizes microtubule networks, inducing cold stability, nocodazole resistance, and tubulin detyrosination on microtubules in transfected cells. We conclude that STOP must play an important role in the generation of microtubule cold stability and in the control of microtubule dynamics in brain.
Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages.
Authors: Hambleton J , Weinstein S L , Lem L , DeFranco A L
Published: July 2002

Activation of macrophages by bacterial lipopolysaccharide (LPS) induces transcription of genes that encode for proinflammatory regulators of the immune response. Previous work has suggested that activation of the transcription factor activator protein 1 (AP-1) is one LPS-induced event that mediates this response. Consistent with this notion, we found that LPS stimulated AP-1-mediated transcription of a transfected reporter gene in the murine macrophage cell line RAW 264.7. As AP-1 activity is regulated in part by activation of the c-Jun N-terminal kinase (JNK), which phosphorylates and subsequently increases the transcriptional activity of c-Jun, we examined whether LPS treatment of macrophages resulted in activation of this kinase. LPS treatment of RAW 264.7 cells, murine bone marrow-derived macrophages, and the human monocyte cell line THP-1 resulted in rapid activation of the p46 and p54 isoforms of JNK. Treatment with wild-type and rough mutant forms of LPS and synthetic lipid A resulted in JNK activation, while pretreatment with the tyrosine kinase inhibitor herbimycin A inhibited this response. Binding of LPS-LPS binding protein (LBP) complexes to CD14, a surface receptor that mediates many LPS responses, was found to be crucial, as pretreatment of THP-1 cells with the monoclonal antibody 60b, which blocks this binding, inhibited JNK activation. These results suggest that LPS activation of JNK in monocyte/macrophage cells is a CD14- and protein tyrosine phosphorylation-dependent event that may mediate the early activation of AP-1 in regulating LPS-triggered gene induction.