bluuebunny/crossref_metadata_2025 huggingface dataset:
Biomimetic macrocyclic receptors for carboxylate anion recognition based on
C
-linked peptidocalix[4]arenes
Authors: Sansone Francesco , Baldini Laura , Casnati Alessandro , Lazzarotto Marcio , Ugozzoli Franco , Ungaro Rocco
Published: July 2002

Two neutral macrobicyclic anion receptors 4 and 6, containing a calix[4]arene in the cone conformation, two
l
-alanine units, and a 2,6-diacylpyridine or a phthaloyl bridge, are described. The x-ray crystal structure of the acetone complexes of the pyridine containing macrocycle 6 shows the four amide NH groups to be in close proximity to the chiral pocket delimited by the pyridine and one aromatic nucleus of the calix[4]arene. This conformation is also the most stable in acetone-
d6
solution, as proven by one- and two-dimensional NMR spectral measurements. Electrospray ionization–MS and
1
H NMR experiments reveal that the two ligands strongly bind carboxylate anions in acetone solution. H-bonding interactions between the carboxylate anions and the amide NH groups, together with π/π stacking, are invoked to explain the efficiency and the selectivity of these anion receptors.
Genetic fidelity under harsh conditions: Analysis of spontaneous mutation in the thermoacidophilic archaeon
Sulfolobus acidocaldarius
Authors: Grogan Dennis W. , Carver Geraldine T. , Drake John W.
Published: July 2002

Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046). However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions. In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes. Both of these conditions are met by the extreme thermoacidophile
Sulfolobus acidocaldarius
. We determined the genomic mutation rate for this species when growing at pH 3.5 and 75°C based on the rate of forward mutation at the
pyrE
gene and the nucleotide changes identified in 101 independent mutants. The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA. The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the
S. acidocaldarius
spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs. The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of
Sulfolobus
spp. It suggests, however, that molecular evolution per genome replication may proceed more slowly in
S. acidocaldarius
than in other DNA-based organisms examined to date.
Gene transcription analysis of
Saccharomyces cerevisiae
exposed to neocarzinostatin protein– chromophore complex reveals evidence of DNA damage, a potential mechanism of resistance, and consequences of prolonged exposure
Authors: Schaus Scott E. , Cavalieri Duccio , Myers Andrew G.
Published: July 2002

The natural product neocarzinostatin (NCS), a protein–small
molecule complex, exhibits potent antiproliferative activity in
mammalian cells but has little apparent effect on the growth of the
unicellular eukaryotic organism,
Saccharomyces
cerevisiae
. Here, we show by whole-genome transcription
profiling experiments that incubation of
S. cerevisiae
with NCS leads to dramatic and wide-ranging modifications in the
expression profile of yeast genes. Approximately 18% of yeast
transcripts are altered by 2-fold or more within 4 h of treatment
with NCS. Analysis of the observed transcription profile provides
evidence that yeast rapidly and continuously overexpress multiple
DNA-damage repair genes during NCS exposure. Perhaps to meet the
energetic requirements of continuous DNA-damage repair, yeast cells
enter respiration upon prolonged exposure to NCS, although grown in
nutrient-rich medium. The NCS protein component is readily transported
into
S. cerevisiae
, as demonstrated by fluorescence
microscopy of yeast treated with fluorescently labeled NCS.
Transcription profiling experiments with neocarzinostatin protein alone
implicate a specific resistance mechanism in yeast that targets the NCS
protein component, one involving the nonclassical export pathway. These
experiments provide a detailed picture of the effects of exposure to
NCS upon yeast and the mechanisms they engage as a response to this
protein–small molecule DNA-damaging agent.
Three retrotransposon families in the genome of
Giardia lamblia
: Two telomeric, one dead
Authors: Arkhipova Irina R. , Morrison Hilary G.
Published: July 2002

Transposable elements inhabiting eukaryotic genomes are generally
regarded either as selfish DNA, which is selectively neutral to the
host organism, or as parasitic DNA, deleterious to the host. Thus far,
the only agreed-upon example of beneficial eukaryotic transposons is
provided by
Drosophila
telomere-associated
retrotransposons, which transpose directly to the chromosome
ends and thereby protect them from degradation. This article reports
the transposon content of the genome of the protozoan
Giardia
lamblia
, one of the earliest-branching eukaryotes. A total of
three non-long terminal repeat retrotransposon families have been
identified, two of which are located at the ends of chromosomes, and
the third one contains exclusively dead copies with multiple internal
deletions, nucleotide substitutions, and frame shifts. No other reverse
transcriptase- or transposase-related sequences were found. Thus, the
entire genome of this protozoan, which is not known to reproduce
sexually, contains only retrotransposons that are either confined to
telomeric regions and possibly beneficial, or inactivated and
completely nonfunctional.
Invasive melanoma in Cdk4-targeted mice
Authors: Sotillo Rocío , García Juan F. , Ortega Sagrario , Martín Javier , Dubus Pierre , Barbacid Mariano , Malumbres Marcos
Published: July 2002

Many human tumors harbor mutations that result in deregulation of Cdk4 activity. Most of these mutations involve overexpression of D-type cyclins and inactivation of INK4 inhibitors. In addition, a mutation in the Cdk4 protein has been described in patients with familial melanoma (Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C.,
et al.
(1995)
Science
269, 1281–1284; Zuo, L., Weger, J., Yang, Q., Goldstein, A. M., Tucker, M. A.,
et al.
(1996)
Nat. Genet.
12, 97–99). This mutation, R24C, renders the Cdk4 protein insensitive to inhibition by INK4 proteins including p16

INK4a

, a major candidate for the melanoma susceptibility locus. Here we show that knock-in mice expressing a Cdk4 R24C allele are highly susceptible to melanoma development after specific carcinogenic treatments. These tumors do not have mutations in the p19

ARF

/p53 pathway, suggesting a specific involvement of the p16

INK4a

/Cdk4/Rb pathway in melanoma development. Moreover, by using targeted mice deficient for other INK4 inhibitors, we show that deletion of p18

INK4c

but not of p15

INK4b

confers proliferative advantage to melanocytic tumor growth. These results provide an experimental scenario to study the role of Cdk4 regulation in melanoma and to develop novel therapeutic approaches to control melanoma progression.
Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination.
Authors: Westerman K A , Leboulch P
Published: July 2002

A procedure of reversible immortalization of primary cells was devised by retrovirus-mediated transfer of an oncogene that could be subsequently excised by site-specific recombination. This study focused on the early stages of immortalization: global induction of proliferation and life span extension of cell populations. Comparative analysis of Cre/LoxP and FLP/FRT recombination in this system indicated that only Cre/LoxP operates efficiently in primary cells. Pure populations of cells in which the oncogene is permanently excised were obtained, following differential selection of the cells. Cells reverted to their preimmortalized state, as indicated by changes in growth characteristics and p53 levels, and their fate conformed to the telomere hypothesis of replicative cell senescence. By permitting temporary and controlled expansion of primary cell populations without retaining the transferred oncogene, this strategy may facilitate gene therapy manipulations of cells unresponsive to exogenous growth factors and make practical gene targeting by homologous recombination in somatic cells. The combination of retroviral transfer and site-specific recombination should also extend gene expression studies to situations previously inaccessible to experimentation.
A general assay for antibody catalysis using acridone as a fluorescent tag.
Authors: Reymond J L , Koch T , Schröer J , Tierney E
Published: July 2002

A simple and highly sensitive catalysis assay is demonstrated based on analyzing reactions with acridonetagged compounds by thin-layer chromatography. As little as 1 pmol of product is readily visualized by its blue fluorescence under UV illumination and identified by its retention factor (Rf). Each assay requires only 10 microliters of solution. The method is reliable, inexpensive, versatile, and immediately applicable in repetitive format for screening catalytic antibody libraries. Three examples are presented: (i) the epoxidation of acridone labeled (S)-citronellol. The pair of stereoisomeric epoxides formed is resolved on the plate, which provides a direct selection method for enantioselective epoxidation catalysts. (ii) Oxidation of acridone-labeled 1-hexanol to 1-hexanal. The activity of horse liver alcohol dehydrogenase is detected. (iii) Indirect product labeling of released aldehyde groups by hydrazone formation with an acridone-labeled hydrazide. Activity of catalytic antibodies for hydrolysis of enol ethers is detected.
Two domains within σ
N
(σ
54
) cooperate for DNA binding
Authors: Cannon W. V. , Chaney M. K. , Wang X.-Y. , Buck M.
Published: July 2002

The σ-N (σ
N
) subunit of the bacterial RNA polymerase is a sequence specific DNA-binding protein. The RNA polymerase holoenzyme formed with σ
N
binds to promoters in an inactive form and only initiates transcription when activated by enhancer-binding positive control proteins. We now provide evidence to show that the DNA-binding activity of σ
N
involves two distinct domains: a C-terminal DNA-binding domain that directly contacts DNA and an adjacent domain that enhances DNA-binding activity. The sequences required for the enhancement of DNA binding can be separated from the sequences required for core RNA polymerase binding. These results provide strong evidence for communication between domains within a transcription factor, likely to be important for the function of σ
N
in enhancer-dependent transcription.
Voltage-controlled gating in a large conductance Ca
2+
-sensitive K
+
channel (hslo)
Authors: Stefani E. , Ottolia M. , Noceti F. , Olcese R. , Wallner M. , Latorre R. , Toro L.
Published: July 2002

Large conductance calcium- and voltage-sensitive K
+
(MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca
2+
favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca
2+
concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4–5 elementary charges.
Independent signals regulate development of primary and secondary follicle structure in spleen and mesenteric lymph node
Authors: Fu Yang-Xin , Huang Guangming , Matsumoto Mitsuru , Molina Hector , Chaplin David D.
Published: July 2002

Lymphotoxin-α-deficient (LT-α−/−) mice manifest congenital absence of lymph nodes (LNs) and Peyer’s patches and disturbed spleen follicle structure. The splenic white pulp areas show loss of discrete T and B lymphocyte zones, of follicular dendritic cell (FDC) clusters, and of germinal centers (GCs). Tumor necrosis factor receptor I-deficient (TNFR-I−/−) mice show similar absence of FDC clusters and GCs but retain segregation of T and B cell zones. Rarely are mesenteric LNs found in LT-α−/−mice. These mesenteric LNs show segregation of T and B cell zones similar to wild-type mice. In contrast, mesenteric LNs in TNFR-I−/−mice manifest grossly disturbed organization of T and B cells. Both LT-α−/−and TNFR-I−/−mice lacked FDC clusters in LNs and spleen. Interestingly, although both LT-α−/−and TNFR-I−/−mice that had been immunized with sheep red blood cells failed to form GCs in the spleen, they both developed GC-like clusters of peanut agglutinin-positive (PNA+) cells in their LNs. Furthermore, when lethally irradiated recombination activating gene (RAG)-1-deficient (RAG-1−/−) mice that had received spleen cells from LT-α−/−mice were immunized with sheep red blood cells, they failed to generate PNA+clusters in the reconstituted spleen but showed robust PNA+clusters in the reconstituted LNs. These data demonstrate that the signals that regulate the development of distinct T and B cell zones as well as the signals that regulate B cell activation to produce clusters of PNA+cells differ between the spleen and LNs.
Human
BRCA1
inhibits growth in yeast: Potential use in diagnostic testing
Authors: Humphrey Jeffrey S. , Salim Ali , Erdos Michael R. , Collins Francis S. , Brody Lawrence C. , Klausner Richard D.
Published: July 2002

Germline-inactivating mutations of
BRCA1
result in a hereditary predisposition to breast and ovarian cancer. Truncating mutations of BRCA1 predispose to cancer and can be ascertained by protein truncation testing or sequencing. However, cancer-predisposing missense mutations of
BRCA1
are difficult to distinguish from polymorphisms by genetic testing methods currently used. Here we show that expression of
BRCA1
or
BRCA1
fused to a
GAL4
activation domain in
Saccharomyces cerevesiae
inhibits growth, resulting in small colonies easily distinguishable from vector-transformed controls. The growth inhibitory effect can be localized to sequences encoding the recently described BRCA1 C-terminal domains. Growth suppression by a BRCA1 fusion protein is not influenced by introduction of neutral polymorphisms but is diminished or abolished by frameshift, nonsense, or disease-associated missense mutations located in the C-terminal 305 amino acids of BRCA1. These observations may permit the functional significance of many
BRCA1
sequence changes to be assessed in yeast. Additionally, the correlation of growth suppression with wild-type forms of BRCA1 suggests that the assay may be capable of detecting functionally conserved interactions between the evolutionarily conserved BRCA1 C-terminal domains and cellular elements found in both human and yeast cells.
Specific complex formation between the type II bare lymphocyte syndrome-associated transactivators CIITA and RFX5
Authors: Scholl Thomas , Mahanta Sanjeev K. , Strominger Jack L.
Published: July 2002

Two of the genes defective in the five complementation groups identified in the class II-negative bare lymphocyte syndrome or corresponding laboratory mutants have been cloned. One gene encodes a protein, RFX5, that is a member of the RFX family of DNA binding proteins. The other, CIITA, encodes a large protein with a defined acidic transcriptional activation domain; this protein does not interact with DNA. Expression plasmids encoding regions of RFX5 fused to the GAL4 DNA binding domain activated transcription from a reporter construct containing GAL4 sites in a cotransfection assay in the Raji human B cell line. However, these plasmids produced transcriptional activity in HeLa cells only in conjunction with interferon γ stimulation, a condition in which expression of both CIITA and class II major histocompatibility complex surface proteins are induced. Furthermore, these plasmids were not active in RJ2.2.5, anin vitromutagenized derivative of Raji in which both copies of CIITA are defective. Transcriptional activation by the RFX5 fusion protein could be restored in RJ2.2.5 by cotransfection with a CIITA expression plasmid. Finally, a direct interaction between RFX5 and CIITA was detected with the yeast two-hybrid and far-Western blot assays. Thus, RFX5 can activate transcription only in cooperation with CIITA. RFX5 and CIITA associate to form a complex capable of activating transcription from class II major histocompatibility complex promoters. In this complex, promoter specificity is determined by the DNA binding domain of RFX5 and the general transcription apparatus is recruited by the acidic activation domain of CIITA.
Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1
Authors: Haque S. Jaharul , Wu Qian , Kammer Winfried , Friedrich Karlheinz , Smith Jonathan M. , Kerr Ian M. , Stark George R. , Williams Bryan R. G.
Published: July 2002

Exposure of cells to protein tyrosine phosphatase (PTP) inhibitors causes an increase in the phosphotyrosine content of many cellular proteins. However, the level at which the primary signaling event is affected is still unclear. We show that Jaks are activated by tyrosine phosphorylation in cells that are briefly exposed to the PTP inhibitor pervanadate (PV), resulting in tyrosine phosphorylation and functional activation of Stat6 (in addition to other Stats). Mutant cell lines that lack Jak1 activity fail to support PV-mediated [or interleukin 4 (IL-4)-dependent] activation of Stat6 but can be rescued by complementation with functional Jak1. The docking sites for both Jak1 and Stat6 reside in the cytoplasmic domain of the IL-4 receptor α-chain (IL-4Rα). The glioblastoma-derived cell lines T98G, GRE, and M007, which do not express the IL-4Rα chain, fail to support Stat6 activation in response to either IL-4 or PV. Complementation of T98G cells with the IL-4Rα restores both PV-mediated and IL-4-dependent Stat6 activation. Murine L929 cells, which do not express the γ common chain of the IL-4 receptor, support PV-mediated but not IL-4-dependent Stat6 activation. Thus, Stat6 activation by PV is an IL-4Rα-mediated, Jak1-dependent event that is independent of receptor dimerization. We propose that receptor-associated constitutive PTP activity functions to down-regulate persistent, receptor-linked kinase activity. Inhibition or deletion of PTP activity results in constitutive activation of cytokine signaling pathways.
Constitutive and impaired signaling of leptin receptors containing the Gln → Pro extracellular domain
fatty
mutation
Authors: White David W. , Wang Yanping , Chua Streamson C. , Morgenstern J. P. , Leibel Rudolph L. , Baumann Heinz , Tartaglia Louis A.
Published: July 2002

Leptin (OB), an adipocyte-secreted circulating hormone, and its
receptor (OB-R) are key components of an endocrine loop that regulates
mammalian body weight. In this report we have analyzed signal
transduction activities of OB-R containing the
fatty
mutation [OB-R(
fa
)], a single amino acid
substitution at position 269 (Gln → Pro) in the OB-R extracellular
domain that results in the obese phenotype of the
fatty
rat. We find that this mutant receptor exhibits both ligand-independent
transcriptional activation via interleukin 6 and hematopoietin receptor
response elements and ligand-independent activation of signal
transducer and activator of transcription (STAT) proteins 1 and 3.
However, OB-R(
fa
) is unable to constitutively
activate STAT5B and is highly impaired for ligand induced activation of
STAT5B compared with OB-R(
wt
). Introduction of the
fatty
mutation into a OB-R/G-CSF-R chimera generates a
receptor with constitutive character that is similar but distinct from
that of OB-R(
fa
). Constitutive mutant
OB-R(
fa
) receptor signaling is repressed by
coexpression of OB-R(
wt
). The implications of an
extracellular domain amino acid substitution generating a cytokine
receptor with a partially constitutive phenotype are discussed both in
terms of the mechanism of OB-R triggering and the biology of the
fatty
rat.
Oncogenic transformation induced by the Qin protein is correlated with transcriptional repression
Authors: Li Jian , Thurm Holger , Chang Hwai Wen , Iacovoni Jason S. , Vogt Peter K.
Published: July 2002

The retroviral oncogene
qin
codes for a protein that belongs to the family of the winged helix transcription factors. The viral Qin protein, v-Qin, differs from its cellular counterpart, c-Qin, by functioning as a stronger transcriptional repressor and a more efficient inducer of tumors. This observation suggests that repression may be important in tumorigenesis. To test this possibility, chimeric proteins were constructed in which the Qin DNA-binding domain was fused to either a strong repressor domain (derived from the
Drosophila
Engrailed protein) or a strong activator domain (from the herpes simplex virus VP16 protein). The chimeric transcriptional repressor, Qin–Engrailed, transformed chicken embryo fibroblasts in culture and induced sarcomas in young chickens. The chimeric activator, Qin–VP16, failed to transform cells
in vitro
or
in vivo
and caused cellular resistance to oncogenic transformation by Qin. These data support the conclusion that the Qin protein induces oncogenic transformation by repressing the transcription of genes which function as negative growth regulators or tumor suppressors.
Tissue-specific expression of herpes simplex virus thymidine kinase gene delivered by adeno-associated virus inhibits the growth of human hepatocellular carcinoma in athymic mice
Authors: Su Hua , Lu Ronghua , Chang Judy C. , Kan Yuet Wai
Published: July 2002

About 70% of hepatocellular carcinomas are known to express α-fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α-fetoprotein enhancer and albumin promoter. We previously demonstrated
in vitro
that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells
in vivo
. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus
in vivo
, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of
in vivo
transduction of tumor cell with rAAV.
Rapid, transient, and highly localized induction of plastidial ω-3 fatty acid desaturase mRNA at fungal infection sites in
Petroselinum crispum
Authors: Kirsch Christoph , Takamiya-Wik Monica , Reinold Susanne , Hahlbrock Klaus , Somssich Imre E.
Published: July 2002

Parsley (
Petroselinum crispum
) plants and suspension-cultured cells have been used extensively for studies of non-host-resistance mechanisms in plant/pathogen interactions. We now show that treatment of cultured parsley cells with a defined peptide elicitor of fungal origin causes rapid and large changes in the levels of various unsaturated fatty acids. While linoleic acid decreased and linolenic acid increased steadily for several hours, comparatively sharp increases in oleic acid followed a biphasic time course. In contrast, the overall level of stearic acid remained unaffected. Using a PCR-based approach, a parsley cDNA was isolated sharing high sequence similarity with ω-3 fatty acid desaturases. Subsequent isolation and characterization of a full-length cDNA enabled its functional identification as a plastid-localized ω-3 fatty acid desaturase by complementation of the
Arabidopsis thaliana fad7/8
double mutant which is low in trienoic fatty acids. ω-3 Fatty acid desaturase mRNA accumulated rapidly and transiently in elicitor-treated cultured parsley cells, protoplasts, and leaves, as well as highly localized around fungal infection sites in parsley leaf buds. These results indicate that unsaturated fatty acid metabolism is yet another component of the highly complex, transcriptionally regulated pathogen defense response in plants.
Mixed-backbone oligonucleotides as second generation antisense oligonucleotides:
In vitro
and
in vivo
studies
Authors: Agrawal Sudhir , Jiang Zhiwei , Zhao Qiuyan , Shaw Denise , Cai Qiuyin , Roskey Allysen , Channavajjala Lakshmi , Saxinger Carl , Zhang Ruiwen
Published: July 2002

Antisense oligonucleotides are being evaluated in clinical trials as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have designed mixed-backbone oligonucleotides (MBOs) that contain phosphorothioate segments at the 3′ and 5′ ends and have a modified oligodeoxynucleotide or oligoribonucleotide segment located in the central portion of the oligonucleotide. Some of these MBOs indicate improved properties compared with phosphorothioate oligodeoxynucleotides with respect to affinity to RNA, RNase H activation, and anti-HIV activity. In addition, more acceptable pharmacological,
in vivo
degradation and pharmacokinetic profiles were obtained with these MBOs.
Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues
Authors: Francis Jonathan W. , Sandrock Alfred W. , Bhide Pradeep G. , Vonsattel Jean-Paul , Brown Robert H.
Published: July 2002

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear “gems,” along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.
Tumors of DNA mismatch repair-deficient hosts exhibit dramatic increases in genomic instability
Authors: Baross-Francis Agnes , Andrew Susan E. , Penney Janice E. , Jirik Frank R.
Published: July 2002

DNA mismatch repair (MMR) deficiency is associated with an increased mutational burden and predisposition to certain malignancies. Relatively little is known, however, about gene-specific mutation frequencies within MMR-deficient primary tumors. Thymic lymphomas from
Msh2
−/−
mice were thus analyzed by using a
lacI
-based transgenic shuttle-phage mutation detection system. All tumors exhibited greatly elevated
lacI
gene mutation frequencies, ranging from 3.2- to 17.4-fold above the ≈15-fold elevations present within normal
Msh2
−/−
thymi. In addition,
lacI
genes harboring multiple changes, including clusters of mutations, were found in thymic tumor DNA. The results suggest that an additional mutator activity, such as an error-prone DNA polymerase, leads to increased genomic instability in these MMR-deficient tumors.
Role of peroxisome proliferator-activated receptor α in disease of pancreatic β cells
Authors: Zhou Yan-Ting , Shimabukuro Michio , Wang May-Yun , Lee Young , Higa Moritake , Milburn Joseph L. , Newgard Christopher B. , Unger Roger H.
Published: July 2002

Expression of peroxisome proliferator-activated receptor α (PPARα) and enzymes of fatty acid (FA) oxidation is markedly reduced in the fat-laden, dysfunctional islets of obese, prediabetic Zucker diabetic fatty (
fa/fa)
rats with mutated leptin receptors (OB-R). Leptin, PPARα/retinoid x receptor ligands, and FA all up-regulate PPARα and enzymes of FA oxidation and stimulate [
3
H]-palmitate oxidation in normal islets but not in islets from
fa/fa
rats. Overexpression of normal OB-R in islets of
fa/fa
rats corrects all of the foregoing abnormalities and reverses the diabetic phenotype. PPARα is a OB-R-dependent factor required for normal fat homeostasis in islet cells.
Ionophores and receptors using cation-π interactions: Collarenes
Authors: Choi Hyuk Soon , Suh Seung Bum , Cho Seung Joo , Kim Kwang S.
Published: July 2002

Cation-π interactions are important forces in molecular recognition by biological receptors, enzyme catalysis, and crystal engineering. We have harnessed these interactions in designing molecular systems with circular arrangement of benzene units that are capable of acting as ionophores and models for biological receptors. [
n
]Collarenes are promising candidates with high selectivity for a specific cation, depending on
n
, because of their structural rigidity and well-defined cavity size. The interaction energies of [
n
]collarenes with cations have been evaluated by using
ab initio
calculations. The selectivity of these [
n
]collarenes in aqueous solution was revealed by using statistical perturbation theory in conjunction with Monte Carlo and molecular dynamics simulations. It has been observed that in [
n
]collarenes the ratio of the interaction energies of a cation with it and the cation with the basic building unit (benzene) can be correlated to its ion selectivity. We find that collarenes are excellent and efficient ionophores that bind cations through cation-π interactions. [6]Collarene is found to be a selective host for Li
+
and Mg
2+
, [8]collarene for K
+
and Sr
2+
, and [10]collarene for Cs
+
and Ba
2+
. This finding indicates that [10]collarene and [8]collarene could be used for effective separation of highly radioactive isotopes,
137
Cs and
90
Sr, which are major constituents of nuclear wastes. More interestingly, collarenes of larger cavity size can be useful in capturing organic cations. [12]Collarene exhibits a pronounced affinity for tetramethylammonium cation and acetylcholine, which implies that it could serve as a model for acetylcholinestrase. Thus, collarenes can prove to be novel and effective ionophores/model-receptors capable of heralding a new direction in molecular recognition and host-guest chemistry.
Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background
Authors: McNamara R. K. , Stumpo D. J. , Morel L. M. , Lewis M. H. , Wakeland E. K. , Blackshear P. J. , Lenox R. H.
Published: July 2002

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous
Macs
mutant mice that exhibit an ≈50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the
Macs
mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCɛ expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the
Macs
mutation did not effect IP-MF length or hippocampal PKCɛ expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the
Macs
mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous
Macs
mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.
Fast repair of O
6
-ethylguanine, but not O
6
-methylguanine, in transcribed genes prevents mutation of H-
ras
in rat mammary tumorigenesis induced by ethylnitrosourea in place of methylnitrosourea
Authors: Engelbergs Jörg , Thomale Jürgen , Galhoff Anke , Rajewsky Manfred F.
Published: July 2002

Differential repair of structurally distinct mutagenic lesions in critical genes may influence the cellular risk of malignant conversion. We have investigated rat mammary tumorigenesis induced by
N
-ethyl-
N
-nitrosourea (EtNU) versus
N
-methyl-
N
-nitrosourea (MeNU) with respect to tumor incidence,
ras
gene mutation, and gene-specific repair. Both carcinogens induced mammary adenocarcinomas at high yield. In mammary epithelia (very low expression of O
6
-alkylguanine-DNA alkyltransferase, MGMT), O
6
-methylguanine (O
6
-MeGua) was eliminated from transcribed (H-
ras
and
β-actin
) and inactive genes (
IgE heavy chain
) at the same slow rate as determined for bulk genomic DNA. The persistence of O
6
-MeGua in DNA correlated with a high frequency of G:C → A:T transition mutations at codon 12 of the H-
ras
gene in MeNU-induced tumors. Repair of O
6
-ethylguanine (O
6
-EtGua), too, was slow in the
IgE heavy chain
gene as in bulk DNA. Contrasting with O
6
-MeGua, however, O
6
-EtGua was removed ≈20 times faster from the active H-
ras
and
β-actin
genes via MGMT-independent mechanism(s). Accordingly, no H-
ras
codon 12 mutations were found in EtNU-induced tumors, and 5- to 8-fold surplus alkyltransferase activity of the mammary epithelia—via a bacterial
ada
transgene—did not significantly counteract tumorigenesis in EtNU-exposed contrary to MeNU-treated animals. Neither MeNU- nor EtNU-induced tumors exhibited mutations at codons 13 and 61 of H-
ras
or codons 12, 13, and 61 of K-
ras
. Fast repair of O
6
-EtGua, but not O
6
-MeGua, in transcribed genes thus prevents mutational activation of H-
ras
when rat mammary carcinogenesis is initiated by EtNU in place of MeNU.
Developmental emergence of different forms of neuromodulation in
Aplysia
sensory neurons
Authors: Marcus Emilie A. , Carew Thomas J.
Published: July 2002

The capacity for neuromodulation and biophysical plasticity is a defining feature of most mature neuronal cell types. In several cases, modulation at the level of the individual neuron has been causally linked to changes in the functional output of a neuronal circuit and subsequent adaptive changes in the organism’s behavioral responses. Understanding how such capacity for neuromodulation develops therefore may provide insights into the mechanisms both of neuronal development and learning and memory. We have examined the development of multiple forms of neuromodulation triggered by a common neurotransmitter, serotonin, in the pleural sensory neurons of
Aplysia californica
. We have found that multiple signaling cascades within a single neuron develop sequentially, with some being expressed only very late in development. In addition, our data suggest a model in which, within a single neuromodulatory pathway, the elements of the signaling cascade are developmentally expressed in a “retrograde” manner with the ionic channel that is modulated appearing early in development, functional elements in the second messenger cascade appearing later, and finally, coupling of the second messenger cascade to the serotonin receptor appearing quite late. These studies provide the characterization of the development of neuromodulation at the level of an identified cell type and offer insights into the potential roles of neuromodulatory processes in development and adult plasticity.
Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFκB
Authors: Cronstein Bruce N. , Montesinos M. Carmen , Weissmann Gerald
Published: July 2002

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin’s inhibition of NFκB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, bothin vitroandin vivo. To determinein vivowhether clinically relevant levels of salicylate act via adenosine, via NFκB, or via the “inflammatory” cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFκB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also providein vivoconfirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFκB.
Interactions between an HMG-1 protein and members of the Rel family
Authors: Brickman Joshua M. , Adam Maryse , Ptashne Mark
Published: July 2002

We show that the
Drosophila
protein DSP1, an HMG-1/2-like protein, binds DNA highly cooperatively with three members of the Rel family of transcriptional regulators (NF-κB, the p50 subunit of NF-κB, and the Rel domain of Dorsal). This cooperativity is apparent with DNA molecules bearing consensus Rel-protein-binding sites and is unaffected by the presence of a negative regulatory element, a sequence previously proposed to be important for mediating repression by these Rel proteins. The cooperativity observed in these DNA-binding assays is paralleled by interactions between protein pairs in the absence of DNA. We also show that in HeLa cells, as assayed by transient transfection, expression of DSP1 increases activation by Dorsal from the
twist
promoter and inhibits that activation from the
zen
promoter, consistent with the previously proposed idea that DSP1 can affect the action of Dorsal in a promoter-specific fashion.
Moe1, a conserved protein in
Schizosaccharomyces pombe
, interacts with a Ras effector, Scd1, to affect proper spindle formation
Authors: Chen Chang-rung , Li Ying-chun , Chen Jing , Hou Ming-chin , Papadaki Piyi , Chang Eric C.
Published: July 2002

In fission yeast, Scd1/Ral1 is a putative guanine nucleotide exchange factor for Cdc42sp and also acts as a Ras1 effector necessary for the regulation of cytoskeleton organization. In this study, we have characterized a protein, Moe1, that binds directly to Scd1. A
moe1
null (Δ) mutant exhibits numerous phenotypes indicative of abnormal microtubule functioning, including an abnormality in the spindle.
moe1
Δ mutants are resistant to microtubule destabilizing agents; moreover,
moe1
Δ rescued the growth defects of tubulin mutants containing unstable microtubules. These results suggest that Moe1 induces instability in microtubules. Biochemical and subcellular localization studies suggest that Moe1 and Scd1 colocalize in the nucleus. Furthermore, loss of function in Scd1 or Ras1 also induced abnormality in the spindle and is synthetically lethal with
moe1
Δ producing cells that lack a detectable spindle. These data demonstrate that Moe1 is a component of the Ras1 pathway necessary for proper spindle formation in the nucleus. Human and nematode Moe1 both can substitute for yeast Moe1, indicating that the function of Moe1 in spindle formation has been conserved substantially during evolution.
Proteinase inhibitor-inducing activity of the prohormone prosystemin resides exclusively in the C-terminal systemin domain
Authors: Dombrowski James E. , Pearce Gregory , Ryan Clarence A.
Published: July 2002

Prosystemin is the 200-amino acid precursor of the 18-amino acid polypeptide defense hormone, systemin. Herein, we report that prosystemin was found to be as biologically active as systemin when assayed for proteinase inhibitor induction in young tomato plants and nearly as active in the alkalinization response in
Lycopersicon esculentum
suspension-cultured cells. Similar to many animal prohormones that harbor multiple signals, the systemin precursor contains five imperfect repetitive domains N-terminal to a single systemin domain. Whether the five repetitive domains contain defense signals has not been established. N-terminal deletions of prosystemin had little effect on its activity in tomato plants or suspension-cultured cells. Deletion of the C-terminal region of prosystemin containing the 18-amino acid systemin domain completely abolished its proteinase inhibitor induction and alkalinization activities. The apoplastic fluid from tomato leaves and the medium of cultured cells were analyzed for proteolytic activity that could process prosystemin to systemin. These experiments showed that proteolytic enzymes present in the apoplasm and medium could cleave prosystemin into large fragments, but the enzymes did not produce detectable levels of systemin. Additionally, inhibitors of these proteolytic enzymes did not affect the biological activity of prosystemin. The cumulative data indicated that prosystemin and/or large fragments of prosystemin can be active inducers of defense responses in both tomato leaves and suspension-cultured cells and that the only region of prosystemin that is responsible for activating the defense response resides in the systemin domain.
Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray
Authors: Johannes Gregg , Carter Mark S. , Eisen Michael B. , Brown Patrick O. , Sarnow Peter
Published: July 2002

Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5′ end- dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5′ noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.
Caenorhabditis elegans
Mediator complexes are required for developmental-specific transcriptional activation
Authors: Kwon Jae Young , Park Jin Mo , Gim Byung Soo , Han Sang Jun , Lee Junho , Kim Young-Joon
Published: July 2002

Mediator proteins are required for transcriptional regulation of most genes in yeast. Mammalian Mediator homologs also function as transcriptional coactivators
in vitro
; however, their physiological role in gene-specific transcription is not yet known. To determine the role of Mediator proteins in the development of complex organisms, we purified putative Mediator complexes from
Caenorhabditis elegans
and analyzed their phenotypes
in vivo
.
C. elegans
Mediator homologs were assembled into two multiprotein complexes. RNA interference assays showed that the CeMed6, CeMed7, and CeMed10/CeNut2 gene products are required for the expression of developmentally regulated genes, but are dispensable for expression of the ubiquitously expressed genes tested in this study. Therefore, the gene-specific function of Mediator as an integrator of transcriptional regulatory signals is evolutionarily conserved and is essential for
C. elegans
development.
Dimethylallyl pyrophosphate is not the committed precursor of isopentenyl pyrophosphate during terpenoid biosynthesis from 1-deoxyxylulose in higher plants
Authors: Arigoni Duilio , Eisenreich Wolfgang , Latzel Christoph , Sagner Silvia , Radykewicz Tanja , Zenk Meinhart H. , Bacher Adelbert
Published: July 2002

Cell cultures of
Catharanthus roseus
were supplied with
[2-
13
C,3-
2
H]-deoxyxylulose or
[2-
13
C,4-
2
H]1-deoxyxylulose. Lutein and
chlorophylls were isolated from the cell mass, and hydrolysis of the
chlorophyll mixtures afforded phytol. Isotope labeling patterns of
phytol and lutein were determined by
2
H NMR and
1
H,
2
H-decoupled
13
C NMR. From the
data it must be concluded that the deuterium atom in position 3 of
deoxyxylulose was incorporated into both isopentenyl pyrophosphate
(IPP) and dimethylallyl pyrophosphate with a rate of 75% (with respect
to the internal
13
C label). The detected stereochemical
signature implies that the label is located preferentially in the
(E)-hydrogen atom of IPP. This preferential labeling, in turn, rules
out dimethylallyl pyrophosphate as the compulsory precursor of IPP. In
the experiment with
[2-
13
C,4-
2
H]1-deoxyxylulose, the
13
C label was efficiently transferred to the terpenoids
whereas the
2
H label was completely washed out, most
probably after IPP formation as a consequence of the isomerization and
elongation process. In addition, the data cast light on the
stereochemical course of the dehydrogenation and cyclization steps
involved in the biosynthesis of lutein.