bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2
Authors: Pfeifer Alexander , Kessler Torsten , Silletti Steve , Cheresh David A. , Verma Inder M.
Published: July 2002

Modulation of the balance between pro- and antiangiogenic factors
holds great promise for the treatment of a broad spectrum of human
disease ranging from ischemic heart disease to cancer. This requires
both the identification of angiogenic regulators and their efficient
delivery to target organs. Here, we demonstrate the use of a
noncatalytic fragment of matrix metalloproteinase 2 (termed
PEX) delivered by lentiviral vectors in different angiogenesis models.
Transduction of human endothelial cells with PEX virus suppressed
endothelial invasion and formation of capillary-like structures without
affecting chemotaxis
in vitro
. Lentiviral delivery of
PEX blocked basic fibroblast growth factor-induced matrix
metalloproteinase 2 activation and angiogenesis on chicken
chorioallantoic membranes. PEX expression also inhibited tumor-induced
angiogenesis and tumor growth in a nude mouse model. Thus, our study
shows that lentiviral vectors can deliver sufficient quantities of
antiangiogenic substances to achieve therapeutic effects
in
vivo
.
DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in
Escherichia coli
Authors: Khodursky Arkady B. , Peter Brian J. , Cozzarelli Nicholas R. , Botstein David , Brown Patrick O. , Yanofsky Charles
Published: July 2002

We investigated the global changes in mRNA abundance in
Escherichia coli
elicited by various perturbations of
tryptophan metabolism. To do so we printed DNA microarrays containing
95% of all annotated
E. coli
ORFs. We determined the
expression profile that is predominantly dictated by the activity of
the tryptophan repressor. Only three operons,
trp
,
mtr
, and
aroH
, exhibited appreciable
expression changes consistent with this profile. The quantitative
changes we observed in mRNA levels for the five genes of the
trp
operon were consistent within a factor of 2, with
expectations based on established Trp protein levels. Several operons
known to be regulated by the TyrR protein,
aroF-tyrA
,
aroL
,
aroP
, and
aroG
, were
down-regulated on addition of tryptophan. TyrR can be activated by any
one of the three aromatic amino acids. Only one operon,
tnaAB
, was significantly activated by the presence of
tryptophan in the medium. We uncovered a plethora of likely indirect
effects of changes in tryptophan metabolism on intracellular mRNA
pools, most prominent of which was the sensitivity of arginine
biosynthetic operons to tryptophan starvation.
Mycobacterium tuberculosissignal transduction system required for persistent infections
Authors: Zahrt Thomas C. , Deretic Vojo
Published: July 2002

It is estimated that nearly 2 billion people currently suffer from latentMycobacterium tuberculosisinfection. Although the key front-line antituberculosis drugs are effective in treating individuals with acute tuberculosis, these drugs are ineffective in eliminatingM. tuberculosisduring the persistent stages of latent infection. Consequently, therapeutics that directly target persistent bacilli are urgently needed. We have conducted a global analysis on a group of regulatory determinants that may play a role inM. tuberculosisvirulence, and identified a two-component response regulator whose expression is required for entrance into and maintenance of persistent infection. Inactivation of this response regulator,Rv0981(termed heremprAfor mycobacterial persistence regulator), affectedM. tuberculosisH37Rv growthin vivoin an organ- and infection stage-specific fashion. These results indicate that two-component systems are important for adaptation of the tubercle bacillus during stages of persistent infection.
Gene-experience interaction alters the cholinergic septohippocampal pathway of mice
Authors: Brooks Andrew I. , Cory-Slechta Deborah A. , Federoff Howard J.
Published: July 2002

Spatial learning requires the septohippocampal pathway. The
interaction of learning experience with gene products to modulate the
function of a pathway may underlie use-dependent plasticity. The
regulated release of nerve growth factor (NGF) from hippocampal
cultures and hippocampus, as well as its actions on cholinergic septal
neurons, suggest it as a candidate protein to interact with a learning
experience. A method was used to evaluate NGF gene-experience
interaction on the septohippocampal neural circuitry in mice. The
method permits brain region-specific expression of a new gene by using
a two-component approach: a virus vector directing expression of
cre
recombinase; and transgenic mice carrying genomic
recombination substrates rendered transcriptionally inactive by a
“floxed” stop cassette. Cre recombinase vector delivery into
transgenic mouse hippocampus resulted in recombination in 30% of
infected cells and the expression of a new gene in those cells. To
examine the interaction of the NGF gene and experience, adult mice
carrying a NGF transgene with a floxed stop cassette (NGFXAT) received
a
cre
recombinase vector to produce localized unilateral
hippocampal NGF gene expression, so-called “activated” mice.
Activated and control nonactivated NGFXAT mice were subjected to
different experiences: repeated spatial learning, repeated rote
performance, or standard vivarium housing. Latency, the time to
complete the learning task, declined in the repeated spatial learning
groups. The measurement of interaction between NGF gene expression and
experience on the septohippocampal circuitry was assessed by counting
retrogradely labeled basal forebrain cholinergic neurons projecting to
the hippocampal site of NGF gene activation. Comparison of all NGF
activated groups revealed a graded effect of experience on the
septohippocampal pathway, with the largest change occurring in
activated mice provided with repeated learning experience. These data
demonstrate that plasticity of the adult spatial learning circuitry can
be robustly modulated by experience-dependent interactions with a
specific hippocampal gene product.
Suppressor T cells regulate the nonanergic cell population that remains after peripheral tolerance is induced to the Mls-1 antigen in T cell receptor Vβ8.1 transgenic mice
Authors: Maeda Hironori , Fujimoto Shigeyoshi , Greene Mark I.
Published: July 2002

We have found suppressor T cells that inhibit the proliferative response of naive CD4+T cells in T cell receptor (TCR) Vβ8.1 transgenic mice rendered tolerantin vivoby inoculation of Mls-1a-positive cells. This suppression was mediated by CD4+T cells but not by CD8+T cells or double-negative (DN) cells, and splenic CD4+T cells from tolerant mice displayed a greater suppression than lymph node CD4+T cells. Cell contact was required for efficient suppression, and known inhibitory cytokines such as IL-4, IL-10, and transforming growth factor β were not involved. Suppressor T cells inhibited IL-2 production by naive CD4+T cells, and the addition of exogenous IL-2 diminished the suppressed activity while having little activity on tolerant T cells. Suppression was abolished by the elimination of CD25+T cells in the tolerant CD4+T cell subset. CD25+CD4+T cells suppressed the proliferative response of the residual fraction of the nonanergic population, namely, 6C10+CD4+T cells still present in the tolerant mice. However, 6C10−CD4+T cells still had reduced reactivity to Mls-1aeven after CD25+CD4+T cells were removed and exogenous IL-2 was added. Suppressor cells appear to affect only residual nonanergic cellsin situ, thereby facilitating the maintenance of the unresponsive statein vivo. These data provide a framework for understanding suppressor T cells and explain the difficulties and variables in defining their activity in other systems, because suppressor T cells apparently control only a small population of nonanergic cells in the periphery and may be viewed as a homeostatic mechanism.
Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells
Authors: Huang Pingbo , Lazarowski Eduardo R. , Tarran Robert , Milgram Sharon L. , Boucher Richard C. , Stutts M. Jackson
Published: July 2002

Physical stimulation of airway surfaces evokes liquid secretion, but the events that mediate this vital protective function are not understood. When cystic fibrosis transmembrane conductance regulator (CFTR) channel activity was used as a functional readout, we found signaling elements compartmentalized at both extracellular and intracellular surfaces of the apical cell membrane that activate apical Cl−conductance in Calu-3 cells. At the outer surface, ATP was released by physical stimuli, locally converted to adenosine, and sensed by A2Badenosine receptors. These receptors couple to G proteins, adenylyl cyclase, and protein kinase A, at the intracellular face of the apical membrane to activate colocalized CFTR. Thus, airways have evolved highly efficient mechanisms to “flush” noxious stimuli from airway surfaces by selective activation of apical membrane signal transduction and effector systems.
Identification of XPR-1, a progesterone receptor required for
Xenopus
oocyte activation
Authors: Tian Jingdong , Kim Sammy , Heilig Elizabeth , Ruderman Joan V.
Published: July 2002

Quiescent full-grown
Xenopus
oocytes remain arrested
at the G
2
/M border of meiosis I until exposed to
progesterone, their natural mitogen. Progesterone triggers rapid,
nontranscriptional responses that lead to the translational activation
of stored mRNAs, resumption of the meiotic cell cycles, and
maturation of the oocyte into a fertilizable egg. It has long been
presumed that progesterone activates the oocyte through a novel
nontranscriptional signaling receptor. Here, we provide evidence that a
conventional transcriptional progesterone receptor cloned from
Xenopus
oocytes, XPR-1, is required for oocyte
activation. Overexpression of XPR-1 through mRNA injection
increases sensitivity to progesterone and accelerates
progesterone-activated cell cycle reentry. Injection of XPR-1 antisense
oligonucleotides blocks the ability of oocytes to respond to
progesterone; these oocytes are rescued by subsequent injection of
XPR-1 or the human progesterone receptor PR-B. Antisense-treated
oocytes can be activated in response to inhibition of protein kinase A,
one of the earliest known changes occurring downstream of progesterone
stimulation. These results argue that the conventional progesterone
receptor also functions as the signaling receptor that is responsible
for the rapid nontranscriptional activation of frog oocytes.
The
Schizosaccharomyces pombe
origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein
Authors: Lee Joon-Kyu , Moon Kyeong-Yeop , Jiang Yun , Hurwitz Jerard
Published: July 2002

The interaction between an origin sequence and the origin recognition complex (ORC), which is highly conserved in eukaryotes, is critical for the initiation of DNA replication. In this report, we have examined the interaction between the
Schizosaccharomyces pombe
(sp) autonomously replicating sequence 1 (
ars1
) and the spORC. For this purpose, we have purified the spORC containing all six subunits, a six-subunit complex containing the N-terminal-deleted spOrc4 subunit (spORC
ΔN-Orc4
), and the spOrc4 subunit by using the baculovirus expression system. Wild-type spORC showed sequence-specific binding to
ars1
, and the spOrc4 protein alone showed the same DNA-binding properties as wild-type spORC. In contrast, the spORC
ΔN-Orc4
and the ΔN-spOrc4p alone did not bind significantly to
ars1.
These findings indicate that the N-terminal domain of the spOrc4 protein that contains multiple AT-hook motifs is essential for the
ars1
-binding activity. DNA-binding competition assays with fragments of
ars1
and DNase I footprinting studies with full-length
ars1
revealed that the spORC interacted with several AT-rich sequence regions of
ars1
. These DNA-binding properties of spORC correlate with the previously determined sequence requirements of the
S. pombe ars1.
These studies indicate that because of its unique Orc4 subunit,
S. pombe
uses a mechanism to recognize its origins different from that used by
Saccharomyces cerevisiae.
Regulation of somatic growth by the p160 coactivator p/CIP
Authors: Wang Zhiyong , Rose David W. , Hermanson Ola , Liu Forrest , Herman Thomas , Wu Wei , Szeto Daniel , Gleiberman Anatoli , Krones Anna , Pratt Katherine , Rosenfeld Ron , Glass Christopher K. , Rosenfeld Michael G.
Published: July 2002

A family of p160 coactivators was initially identified based
on ligand-dependent interactions with nuclear receptors and thought to
function, in part, by recruiting CREB-binding protein/p300 to several
classes of transcription factors. One of the p160 factors,
p/CIP/AIB1, often amplified and overexpressed in breast cancer,
also exhibits particularly strong interaction with CREB-binding
protein/p300. In this manuscript, we report that p/CIP, which
exhibits regulated transfer from cytoplasm to nucleus, is required for
normal somatic growth from embryonic day 13.5 through maturity. Our
data suggest that a short stature phenotype of p/CIP gene-deleted
mice reflect both altered regulation of insulin-like growth factor-1
(IGF-1) gene expression in specific tissues and a cell-autonomous
defect of response to IGF-1, including ineffective transcriptional
activities by several classes of regulated transcription factors under
specific conditions. The actions of p/CIP are therefore required for
full expression of a subset of genes critical for regulating
physiological patterns of somatic growth in mammals.
Genetic analysis of
Nicotiana
pollen-part mutants is consistent with the presence of an
S
-ribonuclease inhibitor at the
S
locus
Authors: Golz John F. , Oh Hae-Young , Su Vanessa , Kusaba Makoto , Newbigin Ed
Published: July 2002

Self-incompatibility (SI) is a genetic mechanism that restricts
inbreeding in flowering plants. In the nightshade family
(Solanaceae) SI is controlled by a single multiallelic
S
locus. Pollen rejection in this system requires the interaction of two
S
locus products: a stylar (S)-RNase and its
pollen counterpart (pollen S).
pollen S
has not yet been
cloned. Our understanding of how this gene functions comes from studies
of plants with mutations that affect the pollen but not the stylar SI
response (pollen-part mutations). These mutations are frequently
associated with duplicated
S
alleles, but the absence of
an obvious additional allele in some plants suggests
pollen
S
can also be deleted. We studied
Nicotiana
alata
plants with an additional
S
allele and
show that duplication causes a pollen-part mutation in several
different genetic backgrounds. Inheritance of the duplication was
consistent with a competitive interaction model in which any two
nonmatching
S
alleles cause a breakdown of SI when
present in the same pollen grain. We also examined plants with presumed
deletions of
pollen S
and found that they instead have
duplications that included
pollen S
but not the S-RNase
gene. This finding is consistent with a bipartite structure for the
S
locus. The absence of
pollen S
deletions in this study and perhaps other studies suggests that
pollen S
might be required for pollen viability,
possibly because its product acts as an S-RNase inhibitor.
Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders.
Authors: Yoshida K , Taga T , Saito M , Suematsu S , Kumanogoh A , Tanaka T , Fujiwara H , Hirata M , Yamagami T , Nakahata T , Hirabayashi T , Yoneda Y , Tanaka K , Wang W Z , Mori C , Shiota K , Yoshida N , Kishimoto T
Published: July 2002

gp130 is a ubiquitously expressed signal-transducing receptor component shared by interleukin 6, interleukin 11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1. To investigate physiological roles of gp130 and to examine pathological consequences of a lack of gp130, mice deficient for gp130 have been prepared. Embryos homozygous for the gp130 mutation progressively die between 12.5 days postcoitum and term. On 16.5 days postcoitum and later, they show hypoplastic ventricular myocardium without septal and trabecular defect. The subcellular ultrastructures in gp130-/- cardiomyocytes appear normal. The mutant embryos have greatly reduced numbers of pluripotential and committed hematopoietic progenitors in the liver and differentiated lineages such as T cells in the thymus. Some gp130-/- embryos show anemia due to impaired development of erythroid lineage cells. These results indicate that gp130 plays a crucial role in myocardial development and hematopoiesis during embryogenesis.
Myosin dynamics in live Dictyostelium cells.
Authors: Moores S L , Sabry J H , Spudich J A
Published: July 2002

Conventional myosin plays a key role in the cytoskeletal reorganization necessary for cytokinesis, migration, and morphological changes associated with development in nonmuscle cells. We have made a fusion between the green fluorescent protein (GFP) and the Dictyostelium discoideum myosin heavy chain (GFP-myosin). The unique Dictyostelium system allows us to test the GFP-tagged myosin for activity both in vivo and in vitro. Expression of GFP-myosin rescues all myosin null cell defects. Additionally, GFP-myosin purified from these cells exhibits the same ATPase activities and in vitro motility as wild-type myosin. GFP-myosin is concentrated in the cleavage furrow during cytokinesis and in the posterior cortex of migrating cells. Surprisingly, GFP-myosin concentration increases transiently in the tips of retracting pseudopods. Contrary to previous thinking, this suggests that conventional myosin may play an important role in the dynamics of pseudopods as well as filopodia, lamellipodia, and other cellular protrusions.
Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.
Authors: DeBernardi M A , Brooker G
Published: July 2002

The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in vivo as an increased FlCRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level.
How cellular slime molds evade nematodes.
Authors: Kessin R H , Gundersen G G , Zaydfudim V , Grimson M
Published: July 2002

We have found a predator-prey association between the social amoeba Dictyostelium discoideum and the free soil living nematode Caenorhabditis elegans. C. elegans feeds on the amoebae and multiplies indefinitely when amoebae are the sole food source. In an environment created from soil, D. discoideum grows and develops, but not in the presence of C. elegans. During development, C. elegans feeds on amoebae until they aggregate and synthesize an extracellular matrix called the slime sheath. After the sheath forms, the aggregate and slug are protected. Adult nematodes ingest Dictyostelium spores, which pass through the gut of the worm without loss of structure and remain viable. Nematodes kill the amoebae but disperse the spores. The sheath that is constructed when the social amoebae aggregate and the spore coats of the individual cells may protect against this predator. Individual amoebae may also protect themselves by secreting compounds that repel nematodes.
Identification of functional domains and evolution of Tc1-like transposable elements.
Authors: Ivics Z , Izsvak Z , Minter A , Hackett P B
Published: July 2002

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.
Interaction of the two cytosolic domains of mammalian adenylyl cyclase.
Authors: Whisnant R E , Gilman A G , Dessauer C W
Published: July 2002

Adenylyl cyclase activity can be reconstituted by simple mixture of the two cytosolic domains of the enzyme after their independent synthesis in Escherichia coli. We have synthesized and purified the C1a domain of type I adenylyl cyclase and the C2 domain of the type II enzyme to assess their interactions with each other and with the activators Gsalpha and forskolin. In the absence of an activator, the fragments associate with low affinity and display low catalytic activity. This basal activity can be stimulated more than 100-fold by either forskolin or activated Gsalpha. Further, the addition of these activators increases the apparent affinity of the fragments for each other. Stimulation of catalysis by Gsalpha and forskolin is synergistic. These data suggest a model wherein either Gsalpha or forskolin enhances association of the other activator with adenylyl cyclase, as well as facilitating the interaction between the C1 and C2 domains of the enzyme.
Bootstrap confidence levels for phylogenetic trees.
Authors: Efron B , Halloran E , Holmes S
Published: July 2002

Evolutionary trees are often estimated from DNA or RNA sequence data. How much confidence should we have in the estimated trees? In 1985, Felsenstein [Felsenstein, J. (1985) Evolution 39, 783-791] suggested the use of the bootstrap to answer this question. Felsenstein's method, which in concept is a straightforward application of the bootstrap, is widely used, but has been criticized as biased in the genetics literature. This paper concerns the use of the bootstrap in the tree problem. We show that Felsenstein's method is not biased, but that it can be corrected to better agree with standard ideas of confidence levels and hypothesis testing. These corrections can be made by using the more elaborate bootstrap method presented here, at the expense of considerably more computation.
Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.
Authors: Mørtz E , O'Connor P B , Roepstorff P , Kelleher N L , Wood T D , McLafferty F W , Mann M
Published: July 2002

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.
Do cyanobacteria swim using traveling surface waves?
Authors: Ehlers K M , Samuel A D , Berg H C , Montgomery R
Published: July 2002

Bacteria that swim without the benefit of flagella might do so by generating longitudinal or transverse surface waves. For example, swimming speeds of order 25 microns/s are expected for a spherical cell propagating longitudinal waves of 0.2 micron length, 0.02 micron amplitude, and 160 microns/s speed. This problem was solved earlier by mathematicians who were interested in the locomotion of ciliates and who considered the undulations of the envelope swept out by ciliary tips. A new solution is given for spheres propagating sinusoidal waveforms rather than Legendre polynomials. The earlier work is reviewed and possible experimental tests are suggested.
Single-copy transgenic mice with chosen-site integration.
Authors: Bronson S K , Plaehn E G , Kluckman K D , Hagaman J R , Maeda N , Smithies O
Published: July 2002

We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.
Translocation of PKN from the cytosol to the nucleus induced by stresses.
Authors: Mukai H , Miyahara M , Sunakawa H , Shibata H , Toshimori M , Kitagawa M , Shimakawa M , Takanaga H , Ono Y
Published: July 2002

Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.
Millipede defense: use of detachable bristles to entangle ants.
Authors: Eisner T , Eisner M , Deyrup M
Published: July 2002

The millipede Polyxenus fasciculatus (Diplopoda; Polyxenida) defends itself against ants by use of a pair of bristle tufts at its rear. When attacked, it wipes the tufts against the ants, thereby causing these to become encumbered by bristles that detach from the tufts. Ants contaminated with bristles desist from their assault. The bristles have grappling hooks at the tip by which they lock onto setae of the ants and barbs along their length by which they interlink. In attempting to rid themselves of bristles, ants may succeed only in further entangling themselves by causing the bristles to become enmeshed. Ants heavily contaminated may remain entangled and die. Most millipedes have chemical defenses; polyxenids, instead, have a mechanical weapon.
Induction of cyclin D1 by simian virus 40 small tumor antigen
Authors: Watanabe Genichi , Howe Alan , Lee Richard J. , Albanese Chris , Shu I-Wei , Karnezis Anthony N. , Zon Leonard , Kyriakis John , Rundell Kathleen , Pestell Richard G.
Published: July 2002

Cell-cycle progression is mediated by a coordinated interaction
between cyclin-dependent kinases and their target proteins including
the pRB and E2F/DP-1 complexes. Immunoneutralization and antisense
experiments have established that the abundance of cyclin D1, a
regulatory subunit of the cyclin-dependent kinases, may be
rate-limiting for G
1
phase progression of the cell cycle.
Simian virus 40 (SV40) small tumor (t) antigen is capable of promoting
G
1
phase progression and augments substantially the
efficiency of SV40 transformation through several distinct domains. In
these studies, small t antigen stimulated cyclin D1 promoter activity
7-fold, primarily through an AP-1 binding site at −954 with additional
contributions from a CRE site at −57. The cyclin D1 AP-1 and CRE sites
were sufficient for activation by small t antigen when linked to an
heterologous promoter. Point mutations of small t antigen between
residues 97–103 that reduced PP2A binding were partially defective in
the induction of the cyclin D1 promoter. These mutations also reduced
activation of MEK1 and two distinct members of the mitogen-activated
protein kinase family, the ERKs (extracellular signal regulated
kinases) and the SAPKs (stress-activated protein kinases), in
transfected cells. Dominant negative mutants of either MEK1, ERK or
SEK1, reduced small t-dependent induction of the cyclin D1 promoter.
SV40 small t induction of the cyclin D1 promoter involves both the ERK
and SAPK pathways that together may contribute to the proliferative and
transformation enhancing activity of small t antigen.
Inhibition of granulocytic differentiation by mNotch1
Authors: Milner Laurie A. , Bigas Anna , Kopan Raphael , Brashem-Stein Carolyn , Bernstein Irwin D. , Martin David I. K.
Published: July 2002

Effective hematopoiesis requires the commitment of pluripotent and multipotent stem cells to distinct differentiation pathways, proliferation and maturation of cells in the various lineages, and preservation of pluripotent progenitors to provide continuous renewal of mature blood cells. While the importance of positive and negative cytokines in regulating proliferation and maturation of hematopoietic cells has been well documented, the factors and molecular processes involved in lineage commitment and self-renewal of multipotent progenitors have not yet been defined. In other developmental systems, cellular interactions mediated by members of theNotchgene family have been shown to influence cell fate determination by multipotent progenitors. We previously described the expression of the humanNotch1homolog,TAN-1, in immature hematopoietic precursors. We now demonstrate that constitutive expression of the activated intracellular domain of mouseNotch1in 32D myeloid progenitors inhibits granulocytic differentiation and permits expansion of undifferentiated cells, findings consistent with the known function ofNotchin other systems.
Costimulatory function and expression of CD40 ligand, CD80, and CD86 in vascularized murine cardiac allograft rejection
Authors: Hancock Wayne W. , Sayegh Mohamed H. , Zheng Xiang-Guang , Peach Robert , Linsley Peter S. , Turka Laurence A.
Published: July 2002

Recent data implicates a role for the CD40–CD40 ligand (CD40L) pathway in graft rejection. One potential mechanism is direct costimulation of T cells through CD40L. Alternatively, the ability of CD40 stimulation to induce CD80 (B7-1) and CD86 (B7-2) expression on antigen-presenting cells (APCs) has led to the hypothesis that the role of CD40–CD40L interactions in transplant rejection might be indirect, i.e., to promote the costimulatory capacity of APCs. Here, we have used a murine vascularized cardiac allograft model to test this hypothesis. Treatment of the recipients with donor splenocytes and a single dose of anti-CD40L mAb induces long-term graft survival (>100 days) in all animals. This is associated with marked inhibition of intragraft Th1 cytokine [interferon γ and interleukin (IL) 2] and IL-12 expression with reciprocal up-regulation of Th2 cytokines (IL-4 and IL-10). In untreated allograft recipients, CD86 is strongly expressed on endothelial cells and infiltrating mononuclear cells of the graft within 24 hr. In contrast, CD80 expression is not seen until 72 hr after engraftment. Anti-CD40L mAb has no detectable effect on CD86 up-regulation, but almost completely abolishes induction of CD80. However, animals treated with anti-CD80 mAb or with a mutated form of CTLA4Ig (which does not bind to CD86) rejected their cardiac allografts, indicating that blockade of CD80 alone does not mediate the graft-prolonging effects of anti-CD40L mAb. These data support the notion that the role of CD40–CD40L in transplant rejection is not solely to promote CD80 or CD86 expression, but rather that this pathway can directly and independently costimulate T cells. These data also suggest that long-term graft survival can be achieved without blockade of either T cell receptor-mediated signals or CD28–CD86 engagement.
Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants
Authors: Flachmann Ralf , Kühlbrandt Werner
Published: July 2002

A chimeric
Lhcb
gene encoding light-harvesting
chlorophyll a/b-binding protein (LHCII) was expressed in transgenic
tobacco plants. To separate native from recombinant LHCII, the protein
was extended by six histidines at its C terminus. Recombinant LHCII was
isolated by detergent-mediated monomerization of pure trimers followed
by affinity-chromatography on Ni
2+
-NTA-agarose (NTA is
nitrilotriacetic acid). Elution with imidazole yielded recombinant
monomers that formed trimers readily after dilution of the detergent
without further
in vitro
manipulations. LHCII subunits
showed the typical chlorophyll a/b ratio at all steps of purification
indicating no significant loss of pigments. Transgenic tobacco
overexpressed amounts of recombinant protein that corresponded to about
0.7% of total LHCII. This yield suggested that expression
in
planta
might be an alternative to the expression of eukaryotic
membrane proteins in yeast. Recombinant LHCII was able to form
two-dimensional crystals after addition of digalactolipids, which
diffracted electrons to 3.6-Å resolution. LHCII carrying a replacement
of Arg-21 with Gln accumulated to only 0.004% of total thylakoid
proteins. This mutant was monomeric in the photosynthetic membrane
probably due to the deletion of the phosphatidylglycerol binding site
and was degraded by the plastidic proteolytic system. Exchange of
Asn-183 with Leu impaired LHCII biogenesis in a similar way presumably
due to the lack of a chlorophyll a binding site.
Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus
Authors: Sorenson Michael D. , Fleischer Robert C.
Published: July 2002

Transpositions of mtDNA sequences to the nuclear genome have been documented in a wide variety of individual taxa, but little is known about their taxonomic frequency or patterns of variation. We provide evidence of nuclear sequences homologous to the mtDNA control region in seven species of diving ducks (tribe Aythyini). Phylogenetic analysis places each nuclear sequence as a close relative of the mtDNA haplotypes of the specie(s) in which it occurs, indicating that they derive from six independent transposition events, all occurring within the last ≈1.5 million years. Relative-rate tests and comparison of intraspecific variation in nuclear and mtDNA sequences confirm the expectation of a greatly reduced rate of evolution in the nuclear copies. By representing mtDNA haplotypes from ancestral populations, nuclear insertions may be valuable in some phylogenetic analyses, but they also confound the accurate determination of mtDNA sequences. In particular, our data suggest that the presumably nonfunctional but more slowly evolving nuclear sequences often will not be identifiable by changes incompatible with function and may be preferentially amplified by PCR primers based on mtDNA sequences from related taxa.
Antiproliferative properties of the USF family of helix-loop-helix transcription factors.
Authors: Luo X , Sawadogo M
Published: July 2002

USF is a family of transcription factors characterized by a highly conserved basic-helix-loop-helix-leucine zipper (bHLH-zip) DNA-binding domain. Two different USF genes, termed USF1 and USF2, are ubiquitously expressed in both humans and mice. The USF1 and USF2 proteins contain highly divergent transcriptional activation domains but share extensive homologies in the bHLH-zip region and recognize the same CACGTG DNA motifs. Although the DNA-binding and transcriptional activities of these proteins have been characterized, the biological function of USF is not well understood. Here, focus- and colony-formation assays were used to investigate the potential involvement of USF in the regulation of cellular transformation and proliferation. Both USF1 and USF2 inhibited the transformation of rat embryo fibroblasts mediated by Ras and c-Myc, a bHLH-zip transcription factor that also binds CACGTG motifs. DNA binding was required but not fully sufficient for inhibition of Myc-dependent transformation by USF, since deletion mutants containing only the DNA-binding domains of USF1 or USF2 produced partial inhibition. While the effect of USF1 was selective for Myc-dependent transformation, wild-type USF2 exerted in addition a strong inhibition of E1A-mediated transformation and a strong suppression of HeLa cell colony formation. These results suggest that members of the USF family may serve as negative regulators of cellular proliferation in two ways, one by antagonizing the transforming function of Myc, the other through a more general growth-inhibitory effect.
In vivo protein-DNA interactions at human DNA replication origin.
Authors: Dimitrova D S , Giacca M , Demarchi F , Biamonti G , Riva S , Falaschi A
Published: July 2002

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basic-helix-loop-helix family, nuclear respiratory factor 1, transcription factor Sp1, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation.
Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells.
Authors: Duncan R L , Kizer N , Barry E L , Friedman P A , Hruska K A
Published: July 2002

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.
Expression of protein encoded by varicella-zoster virus open reading frame 63 in latently infected human ganglionic neurons.
Authors: Mahalingam R , Wellish M , Cohrs R , Debrus S , Piette J , Rentier B , Gilden D H
Published: July 2002

The ganglionic cell type in which varicella-zoster virus (VZV) is latent in humans was analyzed by using antibodies raised against in vitro-expressed VZV open reading frame 63 protein. VZV open reading frame 63 protein was detected exclusively in the cytoplasm of neurons of latently infected human trigeminal and thoracic ganglia. This is, to our knowledge, the first identification of a herpesvirus protein expressed during latency in the human nervous system.
Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.
Authors: Tombline G , Bellizzi D , Sgaramella V
Published: July 2002

In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.
Terminal differentiation of human liposarcoma cells induced by ligands for peroxisome proliferator-activated receptor γ and the retinoid X receptor
Authors: Tontonoz Peter , Singer Samuel , Forman Barry M. , Sarraf Pasha , Fletcher Jonathan A. , Fletcher Christopher D. M. , Brun Regina P. , Mueller Elisabetta , Altiok Soner , Oppenheim Heather , Evans Ronald M. , Spiegelman Bruce M.
Published: July 2002

Induction of terminal differentiation represents a promising therapeutic approach to certain human malignancies. The peroxisome proliferator-activated receptor γ (PPARγ) and the retinoid X receptor α (RXRα) form a heterodimeric complex that functions as a central regulator of adipocyte differentiation. Natural and synthetic ligands for both receptors have been identified. We demonstrate here that PPARγ is expressed at high levels in each of the major histologic types of human liposarcoma. Moreover, primary human liposarcoma cells can be induced to undergo terminal differentiation by treatment with the PPARγ ligand pioglitazone, suggesting that the differentiation block in these cells can be overcome by maximal activation of the PPAR pathway. We further demonstrate that RXR-specific ligands are also potent adipogenic agents in cells expressing the PPARγ/RXRα heterodimer, and that simultaneous treatment of liposarcoma cells with both PPARγ- and RXR-specific ligands results in an additive stimulation of differentiation. Liposarcoma cell differentiation is characterized by accumulation of intracellular lipid, induction of adipocyte-specific genes, and withdrawal from the cell cycle. These results suggest that PPARγ ligands such as thiazolidinediones and RXR-specific retinoids may be useful therapeutic agents for the treatment of liposarcoma.
Experimental surgery to create subgenomes of
Bacillus subtilis
168
Authors: Itaya Mitsuhiro , Tanaka Teruo
Published: July 2002

The 4,188-kb circular genome of
Bacillus subtilis
168 was artificially dissected into two stable circular chromosomes
in vivo
, one being the 3,878-kb main genome and the other the 310-kb subgenome that was recovered as covalently closed circular DNA in CsCl-ethidium bromide ultracentrifugation. The minimal requirements to physically separate the 310-kb DNA segment out of the genome were two interrepeat homologous sequences and an origin of DNA replication between them. The subgenome originated from the 1,255–1,551-kb region of the
B. subtilis
genome was essential for the cell to survive because the subgenome was not lost from the cell. The finding that the
B. subtilis
genome has a potential to be divided and the resulting two replicons stably maintained may shed light on origins and formation mechanisms of giant plasmids or second chromosomes present in many bacteria. Similar excision or its reversal process, i.e., integration of large sized covalently closed circular DNA pieces into the main genome, implies significant roles of subgenomes in the exchange of genetic information and size variation of bacterial genomes in bacterial evolution.