bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R
Authors: Srinivasan Jayashree , Schachner Melitta , Catterall William A.
Published: July 2002

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming α subunit and two smaller auxiliary subunits, β1 and β2. The β subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the β2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R. We investigated the binding of the purified sodium channel and the extracellular domain of the β2 subunit to tenascin-C and tenascin-R
in vitro
. Incubation of purified sodium channels on microtiter plates coated with tenascin-C revealed saturable and specific binding with an apparent
K
d
of ≈15 nM. Glutathione
S
-transferase-tagged fusion proteins containing various segments of tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged extracellular domain of β2 subunits. Both purified sodium channels and the extracellular domain of the β2 subunit bound specifically to fibronectin type III repeats 1–2, A, B, and 6–8 of tenascin-C and fibronectin type III repeats 1–2 and 6–8 of tenascin-R but not to the epidermal growth factor-like domain or the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized sodium channels in these locations.
Down-regulation of
RFL
, the
FLO/LFY
homolog of rice, accompanied with panicle branch initiation
Authors: Kyozuka Junko , Konishi Saeko , Nemoto Keisuke , Izawa Takeshi , Shimamoto Ko
Published: July 2002

FLORICAULA
(
FLO
) of
Antirrhinum
and
LEAFY
(
FLY
) of
Arabidopsis
regulate the
formation of floral meristems. To examine whether same mechanisms
control floral development in distantly related species such as
grasses, we isolated
RFL, FLO-LFY
homolog of rice, and
examined its expression and function. Northern analysis showed that
RFL
is expressed predominantly in very young panicle but
not in mature florets, mature leaves, or roots.
In situ
hybridization revealed that
RFL
RNA was expressed in
epidermal cells in young leaves at vegetative growth stage. After the
transition to reproductive stage,
RFL
RNA was detected
in all layers of very young panicle including the apical meristem, but
absent in the incipient primary branches. As development of branches
proceeds,
RFL
RNA accumulation localized in the
developing branches except for the apical meristems of the branches and
secondary branch primordia. Expression pattern of
RFL
raised a possibility that, unlike
FLO
and
LFY,
RFL
might be involved in panicle branching. Transgenic
Arabidopsis
plants constitutively expressing
RFL
from the cauliflower mosaic virus 35S promoter were
produced to test whether 35S-
RFL
would cause similar
phenotype as observed in 35S-
LFY
plants. In
35S-
RFL
plants, transformation of inflorescence meristem
to floral meristem was rarely observed. Instead, development of
cotyledons, rosette leaves, petals, and stamens was severely affected,
demonstrating that
RFL
function is distinct from that of
LFY
. Our results suggest that mechanisms controlling
floral development in rice might be diverged from that of
Arabidopsis
and
Antirrhinum
.
Importance of anchor genomes for any plant genome project
Authors: Messing Joachim , Llaca Victor
Published: July 2002

Progress in agricultural and environmental technologies is
hampered by a slower rate of gene discovery in plants than animals. The
vast pool of genes in plants, however, will be an important resource
for insertion of genes, via biotechnological procedures, into an array
of plants, generating unique germ plasms not achievable by conventional
breeding. It just became clear that genomes of grasses have evolved in
a manner analogous to Lego blocks. Large chromosome segments have been
reshuffled and stuffer pieces added between genes. Although some
genomes have become very large, the genome with the fewest stuffer
pieces, the rice genome, is the Rosetta Stone of all the bigger grass
genomes. This means that sequencing the rice genome as anchor genome of
the grasses will provide instantaneous access to the same genes in the
same relative physical position in other grasses (e.g., corn and
wheat), without the need to sequence each of these genomes
independently. (
i
) The sequencing of the entire genome
of rice as anchor genome for the grasses will accelerate plant gene
discovery in many important crops (e.g., corn, wheat, and rice) by
several orders of magnitudes and reduce research and development costs
for government and industry at a faster pace. (
ii
) Costs
for sequencing entire genomes have come down significantly. Because of
its size, rice is only 12% of the human or the corn genome, and
technology improvements by the human genome project are completely
transferable, translating in another 50% reduction of the costs.
(
iii
) The physical mapping of the rice genome by a group
of Japanese researchers provides a jump start for sequencing the genome
and forming an international consortium. Otherwise, other countries
would do it alone and own proprietary positions.
23S rRNA positions essential for tRNA binding in ribosomal functional sites
Authors: Bocchetta Maurizio , Xiong Liqun , Mankin Alexander S.
Published: July 2002

rRNA plays an important role in function of peptidyl transferase, the catalytic center of the ribosome responsible for the peptide bond formation. Proper placement of the peptidyl transferase substrates, peptidyl-tRNA and aminoacyl-tRNA, is essential for catalysis of the transpeptidation reaction and protein synthesis. In this report, we define a small set of rRNA nucleotides that are most likely directly involved in binding of tRNA in the functional sites of the large ribosomal subunit. By binding biotinylated tRNA substrates to randomly modified large ribosomal subunits fromEscherichia coliand capturing resulting complexes on the avidin resin, we identified four nucleotides in the large ribosomal subunit rRNA (positions G2252, A2451, U2506, and U2585) whose modifications prevent binding of a peptidyl-tRNA analog in the P site and one residue (U2555) whose modification interferes with transfer of peptidyl moiety to puromycin. These nucleotides represent a subset of positions protected by tRNA analogs from chemical modification and significantly narrow the number of 23S rRNA nucleotides that may be directly involved in tRNA binding in the ribosomal functional sites.
Strand compositional asymmetry in bacterial and large viral genomes
Authors: Mrázek Jan , Karlin Samuel
Published: July 2002

Several bacterial genomes exhibit preference for G over C on the DNA leading strand extending from the origin of replication to the ter-region in the genomes of
Escherichia coli
,
Mycoplasma genitalium
,
Bacillus subtilis
, and marginally in
Haemophilus influenzae
,
Mycoplasma pneumoniae
, and
Helicobacter pylori
. Strand compositional asymmetry is not observed in the cyanobacterium
Synechocystis
sp. genome nor in the archaeal genomes of
Methanococcus jannaschii
,
Methanobacterium thermoautotrophicum
, and
Archaeoglobus fulgidus
. A strong strand compositional asymmetry is observed in β-type but not α- or γ-type human herpesviruses featuring G > C downstream of oriL and C > G upstream of oriL. Dinucleotide relative abundances (i.e., dinucleotide representations normalized by the component nucleotide frequencies) are consonant with respect to the leading and lagging strands. Strand compositional asymmetry may reflect on differences in replication synthesis of the leading versus lagging strand, on differences between template and coding strand associated with transcription-coupled repair mechanisms, on differences in gene density between the two strands, on differences in residue and codon biases in relation to gene function, expression level, or operon organization, or on differences in single or context-dependent base mutational rates. The absence of strand asymmetry in the archaeal genomes may reflect the presence of multiple origins of replication.
Sequestration of the G protein βγ subunit complex inhibits receptor-mediated endocytosis
Authors: Lin Hsin Chieh , Duncan Joseph A. , Kozasa Tohru , Gilman Alfred G.
Published: July 2002

Cell surface receptors that mediate endocytosis cluster into clathrin-coated pits, which pinch off to form vesicles that transport the receptors and their ligands. This multi-step process requires the coordinated action of many factors, including GTP-hydrolyzing proteins such as dynamin and regulators of actin cytoskeleton assembly. We note herein that sequestration of heterotrimeric G protein βγ subunits in intact cells strongly inhibits clathrin-coated pit-mediated endocytosis and causes rearrangement of the actin cytoskeleton. Our results suggest that cells contain a pool of free βγ and that it functions constitutively to permit endocytosis.
A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity
Authors: Baba Masanori , Nishimura Osamu , Kanzaki Naoyuki , Okamoto Mika , Sawada Hidekazu , Iizawa Yuji , Shiraishi Mitsuru , Aramaki Yoshio , Okonogi Kenji , Ogawa Yasuaki , Meguro Kanji , Fujino Masahiko
Published: July 2002

The β-chemokine receptor CCR5 is considered to be an attractive target for inhibition of macrophage-tropic (CCR5-using or R5) HIV-1 replication because individuals having a nonfunctional receptor (a homozygous 32-bp deletion in the CCR5 coding region) are apparently normal but resistant to infection with R5 HIV-1. In this study, we found that TAK-779, a nonpeptide compound with a small molecular weight (Mr531.13), antagonized the binding of RANTES (regulated on activation, normal T cell expressed and secreted) to CCR5-expressing Chinese hamster ovary cells and blocked CCR5-mediated Ca2+signaling at nanomolar concentrations. The inhibition of β-chemokine receptors by TAK-779 appeared to be specific to CCR5 because the compound antagonized CCR2b to a lesser extent but did not affect CCR1, CCR3, or CCR4. Consequently, TAK-779 displayed highly potent and selective inhibition of R5 HIV-1 replication without showing any cytotoxicity to the host cells. The compound inhibited the replication of R5 HIV-1 clinical isolates as well as a laboratory strain at a concentration of 1.6–3.7 nM in peripheral blood mononuclear cells, though it was totally inactive against T-cell line-tropic (CXCR4-using or X4) HIV-1.
Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation
Authors: Rizzuto Gabriella , Cappelletti Manuela , Maione Domenico , Savino Rocco , Lazzaro Domenico , Costa Patrizia , Mathiesen Iacob , Cortese Riccardo , Ciliberto Gennaro , Laufer Ralph , La Monica Nicola , Fattori Elena
Published: July 2002

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 μg of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.
The distribution and copy number of
copia
-like retrotransposons in rice (
Oryza sativa
L.) and their implications in the organization and evolution of the rice genome
Authors: Wang Shiping , Liu Nan , Peng Kaiman , Zhang Qifa
Published: July 2002

We used 22 fragments corresponding to the reverse transcriptase domain of
copia
-like retrotransposons as representatives to study the organization and distribution of these elements in the rice genome. The loci detected by these 22 fragments were assigned to 47 locations in the molecular-linkage map involving all 12 chromosomes. The distributional features of
copia
-like retrotransposons found in the rice genome indicated that (
i
) the loci detected were located mainly in one arm of each chromosome; (
ii
) one fragment usually detected several loci that were mapped to similar locations of different chromosomes; (
iii
) retrotransposons sharing high identity in nucleotide sequences were usually assigned to similar locations of the chromosomes; and (
iv
) concurrences of multiple loci, detected by different fragments, in similar locations or stretches of different chromosomes were common in the rice genome. We also determined that the copy number of
copia
-like retrotransposons in rice genome may be as low as ≈100 per haploid genome. The restricted distribution, along with low copy number, suggested that
copia
-like retrotransposons in rice were relatively inactive during evolution compared with those in other plants. The distributional features of the
copia
-like retrotransposons suggested the existence of possible lineages among the rice chromosomes, which in turn suggested that chromosome duplication and diversification may be a mechanism for the origin and evolution of the rice chromosomes. The information provided by fine mapping of the retroelements in the genetic linkage map may also be useful for gene tagging and molecular cloning.
Mutation, recombination, and incipient speciation of bacteria in the laboratory
Authors: Vulić Marin , Lenski Richard E. , Radman Miroslav
Published: July 2002

Mutations in the DNA mismatch repair system increase mutation and recombination. They may thereby promote the genetic divergence that underlies speciation, after which the reacquisition of a functional repair system may sustain that divergence by creating a barrier to recombination. We tested several lines of
Escherichia coli
, derived from a common ancestor and evolved for 20,000 generations, for their recombination ability. Some lines, but not others, had become mismatch repair-defective mutators during experimental evolution, providing different opportunities for DNA sequence divergence. We knocked out the repair system in lines that had retained this function, and we restored function to those lines that had become defective. We then estimated recombination rates in various crosses between these repair-deficient and -proficient strains. The effect of the mismatch repair system on recombination was greatest in those lines that had evolved nonfunctional repair, indicating they had undergone more sequence divergence and, consequently, were more sensitive to the recombination-inhibiting effect of a functional repair system. These results demonstrate the establishment of an incipient genetic barrier between formerly identical lines, and they support a model in which the mismatch repair system can influence speciation dynamics through its simultaneous effects on mutation and recombination.
Differential acute and chronic responses of tumor necrosis factor-deficient mice to experimental brain injury
Authors: Scherbel Uwe , Raghupathi Ramesh , Nakamura Michio , Saatman Kathryn E. , Trojanowski John Q. , Neugebauer Edmund , Marino Michael W. , McIntosh Tracy K.
Published: July 2002

The present study evaluated behavioral and histopathological outcome after controlled cortical impact (CCI) brain injury in mice deficient in tumor necrosis factor [TNF(−/−)] and their wild-type (wt) littermates. Mice were subjected to CCI brain injury [TNF(−/−),n= 10; wt,n= 10] or served as uninjured controls [TNF(−/−),n= 10; wt,n= 10] and were evaluated for deficits in memory retention at 7 days postinjury. Although both brain-injured wt and TNF(−/−) mice exhibited significant memory dysfunction compared to uninjured controls (P< 0.02), the deficits in memory retention in injured TNF(−/−) mice were significantly less severe than in injured wt mice (P< 0.02). A second group of mice was subjected to CCI brain injury [TNF(−/−),n= 20; wt,n= 20] or served as uninjured controls [TNF(−/−),n= 15; wt,n= 15] and were evaluated over a 4-week period for neurological motor function. In the acute posttraumatic period (48 h postinjury), brain-injured TNF(−/−) mice were significantly less impaired than injured wt mice on composite neuroscore (P< 0.001), rotarod (P< 0.05), and beam balance (P< 0.02) tests. However, wt mice recovered from brain injury by 2–3 weeks postinjury, whereas TNF(−/−) mice continued to demonstrate persistent motor deficits up to 4 weeks postinjury. Histopathological analysis at 2 and 4 weeks postinjury revealed that brain-injured TNF(−/−) mice had significantly more cortical tissue loss than wt mice (P< 0.02). Our results suggest that although the presence of TNF in the acute posttraumatic period may be deleterious, this cytokine may play a role in facilitating long-term behavioral recovery and histological repair after brain injury.
Focal adhesion kinase promotes phospholipase C-γ1 activity
Authors: Zhang Xiaoe , Chattopadhyay Ansuman , Ji Qun-sheng , Owen James D. , Ruest Paul J. , Carpenter Graham , Hanks Steven K.
Published: July 2002

The nonreceptor tyrosine kinase FAK (“focal adhesion kinase”) is a key mediator of integrin signaling events controlling cellular responses to the extracellular matrix, including spreading, migration, proliferation, and survival. Integrin-ligand interactions stimulate FAK tyrosine phosphorylation and activation of FAK signaling functions. Here evidence is presented that the FAK autophosphorylation site Tyr-397 mediates a direct interaction with the C-terminal Src homology 2 domain of phospholipase C (PLC)-γ1 and that this is required for both adhesion-dependent association of the two molecules and increased inositol phosphate production in mouse embryo fibroblasts. Overexpression of FAK and PLC-γ1 in COS-7 cells increases PLC-γ1 enzymatic activity and tyrosine phosphorylation, also dependent on FAK Tyr-397. However, FAK appears incapable of directly phosphorylating PLC-γ1. These observations suggest a role for FAK in recruiting PLC-γ1 to the plasma membrane at sites of cell-matrix adhesion and there promoting its enzymatic activity, possibly by releasing the repression caused by intramolecular interactions of the PLC-γ1 Src homology domains and/or by positioning it for phosphorylation by associated Src-family kinases. These findings expand the known signaling functions of FAK and provide mechanistic insight into integrin-stimulation of PLC-γ1.
NF-κB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes
Authors: Lee Ho H. , Dadgostar Hajir , Cheng Qingwen , Shu Junyan , Cheng Genhong
Published: July 2002

Activation of CD40 is essential for thymus-dependent humoral immune responses and rescuing B cells from apoptosis. Many of the effects of CD40 are believed to be achieved through altered gene expression. In addition to Bcl-x, a known CD40-regulated antiapoptotic molecule, we identified a related antiapoptotic molecule, A1/Bfl-1, as a CD40-inducible gene. Inhibition of the NF-κB pathway by overexpression of a dominant-active inhibitor of NF-κB abolished CD40-induced up-regulation of both the Bfl-1 and Bcl-x genes and also eliminated the ability of CD40 to rescue Fas-induced cell death. Within the upstream promoter region of Bcl-x, a potential NF-κB-binding sequence was found to support NF-κB-dependent transcriptional activation. Furthermore, expression of physiological levels of Bcl-x protected B cells from Fas-mediated apoptosis in the absence of NF-κB signaling. Thus, our results suggest that CD40-mediated cell survival proceeds through NF-κB-dependent up-regulation of Bcl-2 family members.
Growth suppression by Lkb1 is mediated by a G
1
cell cycle arrest
Authors: Tiainen Marianne , Ylikorkala Antti , Mäkelä Tomi P.
Published: July 2002

Germ-line mutations of
LKB1
(
STK11
) lead to Peutz–Jeghers syndrome characterized by gastrointestinal polyps and cancer of different organ systems. The mutations lead to loss or severe impairment of Lkb1 serine/threonine kinase activity. Therefore
LKB1
has been implicated as a tumor suppressor gene, but only a few mutations in the coding exons of
LKB1
have been detected in sporadic tumors. Here, we have identified tumor cell lines with severely reduced mRNA levels and impaired Lkb1 kinase activity. Reintroducing Lkb1 into these cells suppressed cell growth. The Lkb1-mediated growth inhibition was caused by a G
1
cell cycle block and was not detected with several naturally occurring Lkb1 mutants. These results indicate that
LKB1
has functional and specific growth-suppressing activity.
A 21-kDa surface protein of
Mycobacterium leprae
binds peripheral nerve laminin-2 and mediates Schwann cell invasion
Authors: Shimoji Yoshihiro , Ng Vincent , Matsumura Kiichiro , Fischetti Vincent A. , Rambukkana Anura
Published: July 2002

Nerve damage is the hallmark of
Mycobacterium leprae
infection, which results from
M. leprae
invasion of the Schwann cell of the peripheral nervous system. We have recently shown that the laminin-2 isoform, specially the G domain of laminin α2 chain, on the Schwann cell–axon unit serves as an initial neural target for
M. leprae
. However,
M. leprae
surface molecules that mediate bacterial invasion of peripheral nerves are entirely unknown. By using human α2 laminins as a probe, a major 28-kDa protein in the
M. leprae
cell wall fraction that binds α2 laminins was identified. After N-terminal amino acid sequence analysis, PCR-based strategy was used to clone the gene that encodes this protein. Deduced amino acid sequence of this
M. leprae
laminin-binding protein predicts a 21-kDa molecule (ML-LBP21), which is smaller than the observed molecular size in SDS/PAGE. Immunofluorescence and immunoelectron microscopy on intact
M. leprae
with mAbs against recombinant (r) ML-LBP21 revealed that the protein is surface exposed. rML-LBP21 avidly bound to α2 laminins, the rG domain of the laminin-α2 chain, and the native peripheral nerve laminin-2. The role of ML-LBP21 in Schwann cell adhesion and invasion was investigated by using fluorescent polystyrene beads coated with rML-LBP21. Although beads coated with rML-LBP21 alone specifically adhered to and were ingested by primary Schwann cells, these functions were significantly enhanced when beads were preincubated with exogenous α2 laminins. Taken together, the present data suggest that ML-LBP21 may function as a critical surface adhesin that facilitates the entry of
M. leprae
into Schwann cells.
Cell adhesion force microscopy
Authors: Sagvolden G. , Giaever I. , Pettersen E. O. , Feder J.
Published: July 2002

The adhesion forces of cervical carcinoma cells in tissue culture
were measured by using the manipulation force microscope, a novel
atomic force microscope. The forces were studied as a function of time
and temperature for cells cultured on hydrophilic and hydrophobic
polystyrene substrates with preadsorbed proteins. The cells attached
faster and stronger at 37°C than at 23°C and better on hydrophilic
than on hydrophobic substrates, even though proteins adsorb much better
to the hydrophobic substrates. Because cell adhesion serves to control
several stages in the cell cycle, we anticipate that the manipulation
force microscope can help clarify some cell-adhesion related issues.
Genomic sequence, structural organization, molecular evolution, and aberrant rearrangement of promyelocytic leukemia zinc finger gene
Authors: Zhang T. , Xiong H. , Kan L.-X. , Zhang C.-K. , Jiao X.-F. , Fu G. , Zhang Q.-H. , Lu L. , Tong J.-H. , Gu B.-W. , Yu M. , Liu J.-X. , Licht J. , Waxman S. , Zelent A. , Chen E. , Chen S.-J.
Published: July 2002

The promyelocytic leukemia zinc finger gene (
PLZF
) is involved in chromosomal translocation t(11;17) associated with acute promyelocytic leukemia. In this work, a 201-kilobase genomic DNA region containing the entire
PLZF
gene was sequenced. Repeated elements account for 19.83%, and no obvious coding information other than
PLZF
is present over this region.
PLZF
contains six exons and five introns, and the exon organization corresponds well with protein domains. There are at least four alternative splicings (AS-I, -II, -III, and -IV) within exon 1. AS-I could be detected in most tissues tested whereas AS-II, -III, and -IV were present in the stomach, testis, and heart, respectively. Although splicing donor and acceptor signals at exon–intron boundaries for AS-I and exons 1–6 were classical (gt–ag), AS-II, -III, and -IV had atypical splicing sites. These alternative splicings, nevertheless, maintained the ORF and may encode isoforms with absence of important functional domains. In mRNA species without AS-I, there is a relatively long 5′ UTR of 6.0 kilobases. A TATA box and several transcription factor binding sites were found in the putative promoter region upstream of the transcription start site.
PLZF
is a well conserved gene from
Caenorhabditis elegans
to human.
PLZF
paralogous sequences are found in human genome. The presence of two
MLL/PLZF-
like alignments on human chromosome 11q23 and 19 suggests a syntenic replication during evolution. The chromosomal breakpoints and joining sites in the index acute promyelocytic leukemia case with t(11;17) also were characterized, which suggests the involvement of DNA damage-repair mechanism.
Novel neurotrophin-1/B cell-stimulating factor-3: A cytokine of the IL-6 family
Authors: Senaldi Giorgio , Varnum Brian C. , Sarmiento Ulla , Starnes Charlie , Lile Jackson , Scully Sheila , Guo Jane , Elliott Gary , McNinch Jennifer , Shaklee Christine L. , Freeman Daniel , Manu Frank , Simonet W. Scott , Boone Thomas , Chang Ming-Shi
Published: July 2002

We have identified a cytokine of the IL-6 family and named it novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3). NNT-1/BSF-3 cDNA was cloned from activated Jurkat human T cell lymphoma cells. Its sequence predicts a 225-aa protein with a 27-aa signal peptide, a molecular mass of 22 kDa in mature form, and the highest homology to cardiotrophin-1 and ciliary neurotrophic factor. The gene for NNT-1/BSF-3 is on chromosome 11q13. A murine equivalent to NNT-1/BSF-3 also was identified, which shows 96% homology to human NNT-1/BSF-3. NNT-1/BSF-3 mRNA is found mainly in lymph nodes and spleen. NNT-1/BSF-3 induces tyrosine phosphorylation of glycoprotein 130 (gp130), leukemia inhibitory factor receptor β, and signal transducer and activator of transcription 3 in the SK-N-MC human neuroblastoma cells. NNT-1/BSF-3 shows activities typical of IL-6 family members.
In vitro
, it supports the survival of chicken embryo motor and sympathetic neurons. In mice, it induces serum amyloid A, potentiates the induction by IL-1 of corticosterone and IL-6, and causes body weight loss and B cell hyperplasia with serum IgG and IgM increase. NNT-1/BSF-3 is a gp130 activator with B-cell stimulating capability.
Mechanisms underlying kainate receptor-mediated disinhibition in the hippocampus
Authors: Frerking M. , Petersen C. C. H. , Nicoll R. A.
Published: July 2002

Kainate (KA) receptor activation depresses stimulus-evoked γ-aminobutyric acid (GABA-mediated) synaptic transmission onto CA1 pyramidal cells of the hippocampus and simultaneously increases the frequency of spontaneous GABA release through an increase in interneuronal spiking. To determine whether these two effects are independent, we examined the mechanism by which KA receptor activation depresses the stimulus-evoked, inhibitory postsynaptic current (IPSC). Bath application of the α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)/KA receptor agonist KA in the presence of the AMPA receptor antagonist GYKI 53655 caused a large increase in spontaneous GABA release and a coincident depression of the evoked IPSC. The depressant action on the evoked IPSC was reduced, but not abolished, by the GABA
B
receptor antagonist SCH 50911, suggesting that the KA-induced increase in spontaneous GABA release depresses the evoked IPSC through activation of presynaptic GABA
B
receptors. KA had no resolvable effect on the potassium-induced increase in miniature IPSC frequency, suggesting that KA does not act through a direct effect on the release machinery or presynaptic calcium influx. KA caused a decrease in pyramidal cell input resistance, which was reduced by GABA
A
receptor antagonists. KA also caused a reduction in the size of responses to iontophoretically applied GABA, which was indistinguishable from the SCH 50911-resistant, residual depression of the evoked IPSC. These results suggest that KA receptor activation depresses the evoked IPSC indirectly by increasing interneuronal spiking and GABA release, leading to activation of presynaptic GABA
B
receptors, which depress GABA release, and postsynaptic GABA
A
receptors, which increase passive shunting.
Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids
Authors: Lepple-Wienhues Albrecht , Belka Claus , Laun Tilmann , Jekle Andreas , Walter Birgit , Wieland Ulrich , Welz Martina , Heil Luzia , Kun Jutta , Busch Gillian , Weller Michael , Bamberg Michael , Gulbins Erich , Lang Florian
Published: July 2002

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca2+influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca2+entry is lacking in CD95-defectivelprlymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca2+influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.
Transport of torsional stress in DNA
Authors: Nelson Philip
Published: July 2002

It is well known that transcription can induce torsional stress in DNA, affecting the activity of nearby genes or even inducing structural transitions in the DNA duplex. It has long been assumed that the generation of significant torsional stress requires the DNA to be anchored, forming a limited topological domain, because otherwise it would spin almost freely about its axis. Previous estimates of the rotational drag have, however, neglected the role of small natural bends in the helix backbone. We show how these bends can increase the drag several thousandfold relative to prior estimates, allowing significant torsional stress even in linear unanchored DNA. The model helps explain several puzzling experimental results on structural transitions induced by transcription of DNA.
Estrogen inhibits the vascular injury response in estrogen receptor β-deficient female mice
Authors: Karas Richard H. , Hodgin Jeffrey B. , Kwoun Moon , Krege John H. , Aronovitz Mark , Mackey William , Gustafsson Jan Åke , Korach Kenneth S. , Smithies Oliver , Mendelsohn Michael E.
Published: July 2002

The protective effects of estrogen in the cardiovascular system result from both systemic effects and direct actions of the hormone on the vasculature. Two estrogen receptors have been identified, ERα and ERβ. We demonstrated previously that estrogen inhibits the response to vascular injury in both wild-type and ERα-deficient mice, and that ERβ is expressed in the blood vessels of each, suggesting a role for ERβ in the vascular protective effects of estrogen. In the present study, we examined the effect of estrogen administration on mouse carotid arterial injury in ERβ-deficient mice. Surprisingly, in ovariectomized female wild-type and ERβ knockout mice, 17β-estradiol markedly and equally inhibited the increase in vascular medial area and the proliferation of vascular smooth muscle cells after vascular injury. These data demonstrate that ERβ is not required for estrogen-mediated inhibition of the response to vascular injury, and suggest that either of the two known estrogen receptors is sufficient to protect against vascular injury, or that another unidentified estrogen receptor mediates the vascular protective effects of estrogen.
p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting
Authors: Nagaich Akhilesh K. , Zhurkin Victor B. , Durell Stewart R. , Jernigan Robert L. , Appella Ettore , Harrington Rodney E.
Published: July 2002

DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32–36°, whereas wt p53 bends it by 51–57°. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by ≈35° and ≈70°, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53–DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.
Periodical distribution of transcription factor sites in promoter regions and connection with chromatin structure
Authors: Ioshikhes Ilya , Trifonov Edward N. , Zhang Michael Q.
Published: July 2002

Nucleosomes regulate transcriptional initiation when positioned in the promoter area. This may require the transcription factor (TF) sites to be correlated with the nucleosome positions and phased on the nucleosome surface. If this is the case, one would expect a periodical distribution of TF sites in the vicinity of promoters, with the nucleosomal period of 10.1–10.5 bp. We examined the distributions of putative binding sites of 323 different TFs along 1,057 sequences of the Eukaryotic Promoter Database (release 50) [Cavin Perier, R., Junier, T. & Bucher, P. (1998)
Nucleic Acids Res.
26, 353–357] and of 218 TFs on 673 sequences of the Lead Exon Database of human promoter sequences. We obtained a statistically significant overrepresentation of TF sites distributed with the main period of 10.1–10.5 bp in the region −50 to +120 around the transcription start site and in few locations nearby. Correlation of the positioning of the TF sites with the nucleosomes is further reinforced by sequence-directed mapping of the nucleosomes, a method previously developed.
The role of an alternative sigma factor in motility and pilus formation in the cyanobacterium
Synechocystis
sp. strain PCC6803
Authors: Bhaya Devaki , Watanabe Natsu , Ogawa Teruo , Grossman Arthur R.
Published: July 2002

Disruption of a gene for an alternative sigma factor, designated
sigF
, in the freshwater, unicellular cyanobacterium
Synechocystis
sp. strain PCC6803 resulted in a pleiotropic phenotype. Most notably, this mutant lost phototactic movement with a concomitant loss of pili, which are abundant on the surface of wild-type cells. The
sigF
mutant also secreted both high levels of yellow–brown and UV-absorbing pigments and a polypeptide that is similar to a large family of extracellular proteins that includes the hemolysins. Furthermore, the
sigF
mutant had a dramatically reduced level of the transcript from two tandemly arranged
pilA
genes (
sll1694
and
sll1695
), which encode major structural components of type IV pili. Inactivation of these
pilA
genes eliminated phototactic movement, though some pili were still present in this strain. Together, these results demonstrate that SigF plays a critical role in motility via the control of pili formation and is also likely to regulate other features of the cell surface. Furthermore, the data provide evidence that type IV pili are required for phototactic movement in certain cyanobacteria and suggest that different populations of pili present on the
Synechocystis
cell surface may perform different functions.
Engineering precision RNA molecular switches
Authors: Soukup Garrett A. , Breaker Ronald R.
Published: July 2002

Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.
Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane
Authors: Kanamori Takashi , Nishikawa Shuh-ichi , Nakai Masato , Shin Injae , Schultz Peter G. , Endo Toshiya
Published: July 2002

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.
Evolution of microbial diversity during prolonged starvation
Authors: Finkel Steven E. , Kolter Roberto
Published: July 2002

Models of evolutionary processes postulate that new alleles appear in populations through random spontaneous mutation. Alleles that confer a competitive advantage in particular environments are selected and populations can be taken over by individuals expressing these advantageous mutations. We have studied the evolutionary process by using
Escherichia coli
cultures incubated for prolonged periods of time in stationary phase. The populations of surviving cells were shown to be highly dynamic, even after many months of incubation. Evolution proceeded along different paths even when the initial conditions were identical. As cultures aged, the takeovers by fitter mutants were incomplete, resulting in the coexistence of multiple mutant forms and increased microbial diversity. Thus, the study of bacterial populations in stationary phase provides a model system for understanding the evolution of diversity in natural populations.
Polymorphism in RANTES chemokine promoter affects HIV-1 disease progression
Authors: Liu Huanliang , Chao David , Nakayama Emi E. , Taguchi Hitomi , Goto Mieko , Xin Xiaomi , Takamatsu Jun-ki , Saito Hidehiko , Ishikawa Yoshihide , Akaza Tatsuya , Juji Takeo , Takebe Yutaka , Ohishi Takeshi , Fukutake Katsuyuki , Maruyama Yoshikazu , Yashiki Shinji , Sonoda Shunro , Nakamura Tetsuya , Nagai Yoshiyuki , Iwamoto Aikichi , Shioda Tatsuo
Published: July 2002

RANTES (regulated on activation normal T cell expressed and secreted) is one of the natural ligands for the chemokine receptor CCR5 and potently suppressesin vitroreplication of the R5 strains of HIV-1, which use CCR5 as a coreceptor. Previous studies showed that peripheral blood mononuclear cells or CD4+lymphocytes obtained from different individuals had wide variations in their ability to secrete RANTES. These findings prompted us to analyze the upstream noncoding region of the RANTES gene, which contains cis-acting elements involved in RANTES promoter activity, in 272 HIV-1-infected and 193 non-HIV-1-infected individuals in Japan. Our results showed that there were two polymorphic positions, one of which was associated with reduced CD4+lymphocyte depletion rates during untreated periods in HIV-1-infected individuals. This mutation, RANTES−28G, occurred at an allele frequency of ≈17% in the non-HIV-1-infected Japanese population and exerted no influence on the incidence of HIV-1 infection. Functional analyses of RANTES promoter activity indicated that the RANTES−28G mutation increases transcription of the RANTES gene. Taken together, these data suggest that the RANTES−28G mutation increases RANTES expression in HIV-1-infected individuals and thus delays the progression of the HIV-1 disease.
Escherichia coli
genes regulated by cell-to-cell signaling
Authors: Baca-DeLancey Rita R. , South Mary M. T. , Ding Xuedong , Rather Philip N.
Published: July 2002

Utilizing the bicistronic reporter transposon mini-Tn
5
lacZ-tet
/
1,
we have identified
lacZ
fusions to four
Escherichia coli
genes/operons that are strongly activated by the accumulation of self-produced extracellular signals. These fusions were designated
cma9, cma48, cma113,
and
cma114
for
c
onditioned
m
edium
a
ctivated. Each of the
cma
fusions was expressed in a growth phase-dependent manner, and the presence of conditioned medium from a stationary phase
E. coli
culture resulted in the premature activation of these fusions in cells at early to mid-logarithmic phase. The
cma48
and
cma114
fusions were dependent on RpoS for growth phase expression and response to extracellular factors. The extracellular factors that activated the
cma9, cma48,
and
cma114
fusions were produced in both rich complex and defined minimal media. The
cma
fusions were shown to be within the
cysK
(
cma9
)
, astD
(
cma48
),
tnaB
(
cma113
), and
gabT
(
cma114
) genes. These genes function in the uptake, synthesis, or degradation of amino acids that yield pyruvate and succinate.
Phylogeny of a rapidly evolving clade: The cichlid fishes of Lake Malawi, East Africa
Authors: Albertson R. C. , Markert J. A. , Danley P. D. , Kocher T. D.
Published: July 2002

Lake Malawi contains a flock of >500 species of cichlid fish that have evolved from a common ancestor within the last million years. The rapid diversification of this group has been attributed to morphological adaptation and to sexual selection, but the relative timing and importance of these mechanisms is not known. A phylogeny of the group would help identify the role each mechanism has played in the evolution of the flock. Previous attempts to reconstruct the relationships among these taxa using molecular methods have been frustrated by the persistence of ancestral polymorphisms within species. Here we describe results from a DNA fingerprinting technique that overcomes this problem by examining thousands of polymorphisms distributed across the genome. The resulting dendrogram averages the evolutionary history of thousands of genes and should accurately reflect the evolutionary history of these species. Our tree resolves relationships among closely related Lake Malawi cichlids and provides insights into the pattern of speciation in this group. We demonstrate that adaptive divergence in trophic morphology played an important role during the early history of the lake. Subsequent species diversity has arisen with little change in trophic morphology, which suggests that other forces are responsible for the continued speciation of these fishes.
Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages
Authors: Lee Benhur , Sharron Matthew , Montaner Luis J. , Weissman Drew , Doms Robert W.
Published: July 2002

CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2- to 5-fold between individuals. CCR5 was expressed at much higher levels in CD4+/CD45RO+/CD62L-true memory cells compared with CD4+/CD45RO+/CD62L+ cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitromaturation of PBDC resulted in median 3- and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from ≈5,000 to ≈50,000 ABS) whereas granulocyte-macrophage colony-stimulating factor caused a marked decrease of CXCR4 (from ≈5,000 ABS to <500) while up-regulating CCR5 expression (from ≈5,000 to ≈20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms.