bluuebunny/crossref_metadata_2025 huggingface dataset:
Crosstalk among stress responses in plants: Pathogen defense overrides UV protection through an inversely regulated ACE/ACE type of light-responsive gene promoter unit
Authors: Logemann Elke , Hahlbrock Klaus
Published: July 2002

Plants often have to cope with two or more environmental hazards simultaneously. Such coincidences require instantaneous decisions on relative severity and consequential crosstalk between the respective signaling cascades. Among the frequently encountered threats are pathogen infections and UV irradiation, both of which trigger specifically targeted defense responses by means of changes in gene transcription rates. In
Petroselinum crispum
, pathogen defense has been shown to be associated with extensive metabolic reprogramming, including strong repression of the UV-protective flavonoid biosynthetic pathway. Here we show that one of the involved genes, encoding acyl-CoA oxidase, responds positively to UV light and negatively to a pathogen-derived elicitor through an inversely regulated promoter unit consisting of two almost identical ACGT-containing elements (ACEs). This unit, when either introduced into an unrelated promoter or generated by mutation of a differently composed unit, confers the same type of response pattern on the recipient genes, confirming its general functionality at a convergence site of two largely distinct signaling pathways. Similarly large, rapid, and partly inverse effects of UV light and elicitor were observed for several mRNAs encoding common plant regulatory factors (CPRFs) that exhibit distinct dimerization and DNA-binding properties. This striking coincidence suggests a major role of common plant regulatory factors in mediating the apparent switch in the function of ACGT-containing elements from positive UV light to negative elicitor or pathogen responsiveness.
Selective interaction between leptin and insulin signaling pathways in a hepatic cell line
Authors: Szanto Ildiko , Kahn C. Ronald
Published: July 2002

Leptin is a 16-kDa hormone secreted by adipocytes and plays an important role in control of feeding behavior and energy expenditure. In obesity, circulating levels of leptin and insulin are high because of the presence of increased body fat mass and insulin resistance. Recent reports have suggested that leptin can act through some of the components of the insulin signaling cascade, such as insulin receptor substrates (IRS-1 and IRS-2), phosphatidylinositol 3-kinase (PI 3-kinase), and mitogen-activated protein kinase, and can modify insulin-induced changes in gene expression
in vitro
and
in vivo
. Well differentiated hepatoma cells (Fao) possess both the long and short forms of the leptin receptor and respond to leptin with a stimulation of
c-fos
gene expression. In Fao cells, leptin alone had no effects on the insulin signaling pathway, but leptin pretreatment transiently enhanced insulin-induced tyrosine phosphorylation and PI 3-kinase binding to IRS-1, while producing an inhibition of tyrosine phosphorylation and PI 3-kinase binding to IRS-2. Leptin alone also induced serine phosphorylation of Akt and glycogen synthase kinase 3 but to a lesser extent than insulin, and the combination of these hormones was not additive. These results suggest complex interactions between the leptin and insulin signaling pathways that can potentially lead to differential modification of the metabolic and mitotic effects of insulin exerted through IRS-1 and IRS-2 and the downstream kinases that they activate.
Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast
Authors: Egner Alexander , Jakobs Stefan , Hell Stefan W.
Published: July 2002

By introducing beam-scanning multifocal multiphoton 4Pi-confocal microscopy, we have attained fast fluorescence imaging of live cells with axial super resolution. Rapid scanning of up to 64 pairs of interfering high-angle fields and subsequent confocal detection enabled us to perform three to five times finer optical sectioning than confocal microscopy. In conjunction with nonlinear image restoration, we demonstrate, to our knowledge for the first time, three-dimensional imaging of live eukaryotic cells at an equilateral resolution of ≈100 nm. This imaging mode allowed us to reveal the morphology and size of the green fluorescent protein-labeled mitochondrial compartment of live
Saccharomyces cerevisiae
(bakers' yeast) growing on different carbon sources. Our studies show that mitochondria of cells grown on medium containing glycerol as the only carbon source, as opposed to glucose-grown cells, exhibit a strongly branched tubular reticulum. We determine the average tubular diameter and find that it increases from 339 ± 5 nm to 360 ± 4 nm when changing from glucose to glycerol, that is, from a fermentable to a nonfermentable carbon source. Moreover, this change is associated with a 2.8-fold increase of the surface of the reticulum, resulting in an average increase in volume of the mitochondrial compartment by a factor of 3.0 ± 0.2.
Selective imaging of presynaptic activity in the mouse olfactory bulb shows concentration and structure dependence of odor responses in identified glomeruli
Authors: Fried Hans U. , Fuss Stefan H. , Korsching Sigrun I.
Published: July 2002

More chemicals can be smelled than there are olfactory receptors for them, necessitating a combinatorial representation by somewhat broadly tuned receptors. To understand the perception of odor quality and concentration, it is essential to establish the nature of the receptor repertoires that are activated by particular odorants at particular concentrations. We have taken advantage of the one-to-one correspondence of glomeruli and olfactory receptor molecules in the mouse olfactory bulb to analyze the tuning properties of a major receptor population by high resolution calcium imaging of odor responses selectively in the presynaptic compartment of glomeruli. We show that eighty different olfactory receptors projecting to the dorsal olfactory bulb respond to high concentrations of aldehydes with limited specificity. Varying ensembles of about 10 to 20 receptors encode any particular aldehyde at low stimulus concentrations with high specificity. Even normalized odor response patterns are markedly concentration dependent, caused by pronounced differences in affinity within the aldehyde receptor repertoire.
Molecular evolution of the HoxA cluster in the three major gnathostome lineages
Authors: Chiu Chi-hua , Amemiya Chris , Dewar Ken , Kim Chang-Bae , Ruddle Frank H. , Wagner Günter P.
Published: July 2002

The duplication of Hox clusters and their maintenance in a lineage has a prominent but little understood role in chordate evolution. Here we examined how Hox cluster duplication may influence changes in cluster architecture and patterns of noncoding sequence evolution. We sequenced the entire duplicated HoxAa and HoxAb clusters of zebrafish (
Danio rerio
) and extended the 5′ (posterior) part of the HoxM (HoxA-like) cluster of horn shark (
Heterodontus francisci
) containing the
hoxa11
and
hoxa13
orthologs as well as intergenic and flanking noncoding sequences. The duplicated HoxA clusters in zebrafish each house considerably fewer genes and are dramatically shorter than the single HoxA clusters of human and horn shark. We compared the intergenic sequences of the HoxA clusters of human, horn shark, zebrafish (Aa, Ab), and striped bass and found extensive conservation of noncoding sequence motifs, i.e., phylogenetic footprints, between the human and horn shark, representing two of the three gnathostome lineages. These are putative cis-regulatory elements that may play a role in the regulation of the ancestral HoxA cluster. In contrast, homologous regions of the duplicated HoxAa and HoxAb clusters of zebrafish and the HoxA cluster of striped bass revealed a striking loss of conservation of these putative cis-regulatory sequences in the 3′ (anterior) segment of the cluster, where zebrafish only retains single representatives of group 1, 3, 4, and 5 (HoxAa) and group 2 (HoxAb) genes and in the 5′ part of the clusters, where zebrafish retains two copies of the group 13, 11, and 9 genes, i.e., AbdB-like genes. In analyzing patterns of cis-sequence evolution in the 5′ part of the clusters, we explicitly looked for evidence of complementary loss of conserved noncoding sequences, as predicted by the duplication-degeneration-complementation model in which genetic redundancy after gene duplication is resolved because of the fixation of complementary degenerative mutations. Our data did not yield evidence supporting this prediction. We conclude that changes in the pattern of cis-sequence conservation after Hox cluster duplication are more consistent with being the outcome of adaptive modification rather than passive mechanisms that erode redundancy created by the duplication event. These results support the view that genome duplications may provide a mechanism whereby master control genes undergo radical modifications conducive to major alterations in body plan. Such genomic revolutions may contribute significantly to the evolutionary process.
A functional chaperone triad on the yeast ribosome
Authors: Gautschi Matthias , Mun Andrej , Ross Suzanne , Rospert Sabine
Published: July 2002

The chaperones RAC (ribosome-associated complex), consisting of Ssz1p and zuotin, and Ssb1/2p are associated with ribosomes of yeast. Ssb1/2p was previously shown to form a crosslink product to polypeptides trapped in ribosome-nascent chain complexes (RNCs)
in vitro
. Here we show that an efficient crosslink of the nascent chain to Ssb1/2p depends on the presence of functional RAC. The crosslink to Ssb1/2p was significantly diminished if (
i
) RAC was removed from RNCs: a process reversed by addition of purified RAC; (
ii
) RAC carried a mutation in the J-domain of zuotin, leading to its inactivation
in vivo
; (
iii
) RAC's Ssz1p subunit was absent because RNCs were generated in a Δ
ssz1
-derived translation extract.
In vivo
the same specific set of growth defects caused by the absence of any of the three chaperones was also displayed by a Δ
ssb1
/
2
Δ
ssz1
Δ
zuo1
strain. The combination of
in vitro
and
in vivo
data supports a model in which Ssb1/2p, Ssz1p, and zuotin act in concert on nascent chains while they are being synthesized.
Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression
Authors: Nicot Arnaud , DiCicco-Bloom Emanuel
Published: July 2002

Although neurogenesis in the embryo proceeds in a region- or
lineage-specific fashion coincident with neuropeptide expression, a
regulatory role for G protein-coupled receptors (GPCR) remains
undefined. Pituitary adenylate cyclase activating polypeptide (PACAP)
stimulates sympathetic neuroblast proliferation, whereas the peptide
inhibits embryonic cortical precursor mitosis. Here, by using ectopic
expression strategies, we show that the opposing mitogenic effects of
PACAP are determined by expression of PACAP receptor splice isoforms
and differential coupling to the phospholipase C (PLC) pathway, as
opposed to differences in cellular context. In embryonic day 14 (E14)
cortical precursors transfected with the hop receptor variant, but not
cells transfected with the short variant, PACAP activates the PLC
pathway, increasing intracellular calcium and eliciting translocation
of protein kinase C. Ectopic expression of the hop variant in cortical
neuroblasts transforms the antimitotic effect of PACAP into a
promitogenic signal. Furthermore, PACAP promitogenic effects required
PLC pathway function indicated by antagonist U-73122 studies in
hop-transfected cortical cells and native sympathetic neuroblasts.
These observations highlight the critical role of lineage-specific
expression of GPCR variants in determining mitogenic signaling in
neural precursors.
Origin ofsphinx, a young chimeric RNA gene inDrosophilamelanogaster
Authors: Wang Wen , Brunet Frédéric G. , Nevo Eviatar , Long Manyuan
Published: July 2002

Non-protein-coding RNA genes play an important role in various biological processes. How new RNA genes originated and whether this process is controlled by similar evolutionary mechanisms for the origin of protein-coding genes remains unclear. A young chimeric RNA gene that we termsphinx (spx)provides the first insight into the early stage of evolution of RNA genes.spxoriginated as an insertion of a retroposed sequence of the ATP synthase chain F gene at the cytological region 60DB since the divergence ofDrosophila melanogasterfrom its sibling species 2–3 million years ago. This retrosequence, which is located at 102F on the fourth chromosome, recruited a nearby exon and intron, thereby evolving a chimeric gene structure. This molecular process suggests that the mechanism of exon shuffling, which can generate protein-coding genes, also plays a role in the origin of RNA genes. The subsequent evolutionary process ofspxhas been associated with a high nucleotide substitution rate, possibly driven by a continuous positive Darwinian selection for a novel function, as is shown in its sex- and development-specific alternative splicing. To test whetherspxhas adapted to different environments, we investigated its population genetic structure in the unique “Evolution Canyon” in Israel, revealing a similar haplotype structure inspx, and thus similar evolutionary forces operating onspxbetween environments.
From intestine to muscle: Nuclear reprogramming through defective cloned embryos
Authors: Byrne J. A. , Simonsson S. , Gurdon J. B.
Published: July 2002

Nuclear transplantation is one of the very few ways by which the genetic content and capacity for nuclear reprogramming can be assessed in individual cells of differentiated somatic tissues. No more than 6% of the cells of differentiated tissues have thus far been shown to have nuclei that can be reprogrammed to elicit the formation of unrelated cell types. In Amphibia, about 25% of such nuclear transfers form morphologically abnormal partial blastulae that die within 24 h. We have investigated the genetic content and capacity for reprogramming of those nuclei that generate partial blastulae, using as donors the intestinal epithelium cells of feeding
Xenopus
larvae. We have analyzed single nuclear transplant embryos obtained directly from intestinal tissue, thereby avoiding any genetic or epigenetic changes that might accumulate during cell culture. The expression of the intestine-specific gene intestinal fatty acid binding protein is extinguished by at least 10
4
times, within a few hours of nuclear transplantation. At the same time several genes that are normally expressed only in early embryos are very strongly activated in nuclear transplant embryos, but to an unregulated extent. Remarkably, cells from intestine-derived partial blastulae, when grafted to normal host embryos, contribute to several host tissues and participate in the normal 100-fold increase in axial muscle over several months. Thus, cells of defective cloned embryos unable to survive for more than 1 day can be reprogrammed to participate in new directions of differentiation and to maintain indefinite growth, despite the abnormal expression of early genes.
Characterization of a high-voltage-activated
I
A
current with a role in spike timing and locomotor pattern generation
Authors: Hess Dietmar , El Manira Abdeljabbar
Published: July 2002

Transient A-type K
+
channels
(
I
A
) in neurons have been implicated in the
delay of the spike onset and the decrease in the firing frequency. Here
we have characterized biophysically and pharmacologically an
I
A
current in lamprey locomotor network
neurons that is activated by suprathreshold depolarization and is
specifically blocked by catechol at 100 μM. The biophysical
properties of this current are similar to the mammalian Kv3.4 channel.
The role of the
I
A
current both in single
neuron firing and in locomotor pattern generation was analyzed. The
I
A
current facilitates Na
+
channel recovery from inactivation and thus sustains repetitive firing.
The role of the
I
A
current in motor pattern
generation was examined by applying catechol during fictive locomotion
induced by
N
-methyl-
d
-aspartate. Blockade of
this current increased the locomotor burst frequency and decreased the
firing of motoneurons. Although an alternating motor pattern could
still be generated, the cycle duration was less regular, with ventral
roots bursts failing on some cycles. Our results thus provide insights
into the contribution of a high-voltage-activated
I
A
current to the regulation of firing
properties and motor coordination in the lamprey spinal cord.
Differential roles for NSF and GRIP/ABP in AMPA receptor cycling
Authors: Braithwaite Steven P. , Xia Houhui , Malenka Robert C.
Published: July 2002

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) stability and movement at synapses are important factors controlling synaptic strength. Here, we study the roles of proteins [
N
-ethylmaleimide-sensitive fusion protein (NSF), glutamate receptor AMPAR binding protein (ABP)-interacting protein (GRIP)/(ABP), and protein interacting with C-kinase-1 (PICK1) that interact with the GluR2 subunit in the control of the surface expression and cycling of AMPARs. Epitope-tagged GluR2 formed functional receptors that exhibited targeting to synaptic sites. Constructs in which binding to NSF, PDZ proteins (GRIP/ABP and PICK1), or GRIP/ABP alone was eliminated each exhibited normal surface targeting and constitutive cycling. The lack of NSF binding, however, resulted in receptors that were endocytosed to a greater extent than wild-type receptors in response to application of AMPA or
N
-methyl-
d
-aspartate (NMDA). Conversely, the behavior of the GluR2 mutants incapable of binding to GRIP/ABP suggests that these PDZ proteins play a role in the stabilization of an intracellular pool of AMPARs that have been internalized on stimulation, thus inhibiting their recycling to the synaptic membrane. These results provide further evidence for distinct functional roles of GluR2-interacting proteins in AMPAR trafficking.
Identification of β-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA
Authors: Olbrot Martin , Rud Jonathan , Moss Larry G. , Sharma Arun
Published: July 2002

Of the three critical enhancer elements that mediate β-cell-specific and glucose-responsive expression of the insulin gene, only the identity of the transcription factor binding to the RIPE3b element (RIPE3b1) has remained elusive. Using a biochemical purification approach, we have identified the RIPE3b1 factor as a
m
ammalian homologue of avian
MafA
/L-Maf (mMafA). The avian MafA is a cell-type determination factor that expressed ectopically can trigger lens differentiation program, but no mammalian homologue of avian MafA has previously been identified. Here, we report cloning of the human
mafA
(hMafA) and demonstrate that it can specifically bind the insulin enhancer element RIPE3b and activate insulin-gene expression. In addition, mMafA has a very restrictive cellular distribution and is selectively expressed in pancreatic β but not in α cells. We suggest that mMafA has an essential role in the function and differentiation of β-cells and thus may be associated with the pathophysiological origins of diabetes.
Clara cell impact in air-side activation of CFTR in small pulmonary airways
Authors: Kulaksiz Hasan , Schmid Andreas , Hönscheid Matthias , Ramaswamy Annette , Cetin Yalcin
Published: July 2002

The Clara cells are nonciliated, nonmucous, secretory cells containing characteristic peptidergic granules; they constitute up to 80% of the epithelial cell population of the distal airways. Despite this exposed histotopology and abundance within the terminal airways where fluid secretion is of pivotal importance, the functional role of the Clara cells remained poorly understood. At the transcriptional, translational, and cellular levels, we provide evidence that the Clara cells are well equipped with the bioactive peptide guanylin and proteins of the cGMP-signaling system including guanylate cyclase C, cGMP-dependent protein kinase II, and cystic fibrosis transmembrane conductance regulator (CFTR) together with the two CFTR scaffolding proteins EBP50/NHERF and E3KARP/NHERF-2 that are essential for proper function of CFTR. Guanylin was localized to secretory granules underneath the apical membrane of Clara cells and was, in addition, detected in high concentrations in bronchoalveolar lavage fluid, predicting release of the peptide luminally into the bronchiolar airways. On the other hand, the guanylin-receptor guanylate cyclase C, CFTR, and proteins linked to CFTR activation and function were all confined to the adluminal membrane of Clara cells, implicating an intriguing air-side route of action of guanylin. Whole-cell patch-clamp recordings in the Clara cell line H441 revealed that guanylin activates CFTR Cl
−
conductance via the cGMP but not the cAMP-signaling pathway. Hence, in the critical location of distal airways
in situ
, the Clara cells may play the outstanding role of CFTR-dependent regulation of epithelial electrolyte/water secretion through a sophisticated paracrine/luminocrine mode of guanylin-induced CFTR activation.
The origin of intermediary metabolism
Authors: Morowitz Harold J. , Kostelnik Jennifer D. , Yang Jeremy , Cody George D.
Published: July 2002

The core of intermediary metabolism in autotrophs is the citric acid cycle. In a certain group of chemoautotrophs, the reductive citric acid cycle is an engine of synthesis, taking in CO
2
and synthesizing the molecules of the cycle. We have examined the chemistry of a model system of C, H, and O that starts with carbon dioxide and reductants and uses redox couples as the energy source. To inquire into the reaction networks that might emerge, we start with the largest available database of organic molecules, Beilstein on-line, and prune by a set of physical and chemical constraints applicable to the model system. From the 3.5 million entries in Beilstein we emerge with 153 molecules that contain all 11 members of the reductive citric acid cycle. A small number of selection rules generates a very constrained subset, suggesting that this is the type of reaction model that will prove useful in the study of biogenesis. The model indicates that the metabolism shown in the universal chart of pathways may be central to the origin of life, is emergent from organic chemistry, and may be unique.
Interactions between heterologous forms of prion protein: Binding, inhibition of conversion, and species barriers
Authors: Horiuchi Motohiro , Priola Suzette A. , Chabry Joëlle , Caughey Byron
Published: July 2002

The self-induced formation of the disease-associated, protease-resistant prion protein (PrP-res) from the normal protease-sensitive isoform (PrP-sen) appears to be a key event in the pathogenesis of transmissible spongiform encephalopathies. The amino acid sequence specificity of PrP-res formation correlates with, and may account for, the species specificity in transmission of transmissible spongiform encephalopathy agents
in vivo
. To analyze the mechanism controlling the sequence specificity of PrP-res formation, we compared the binding of PrP-sen to PrP-res with its subsequent acquisition of protease resistance by using cell-free systems consisting of heterologous versus homologous mouse and hamster PrP isoforms. Our studies showed that heterologous PrP-sen can bind to PrP-res with little conversion to the protease-resistant state and, in doing so, can interfere with the conversion of homologous PrP-sen. The interference occurred with molar ratios of homologous to heterologous PrP-sen molecules as low as 1:1. The interference was due primarily to the inhibition of conversion, but not the binding, of the homologous PrP-sen to PrP-res. The results provide evidence that the sequence specificity of PrP-res formation in this model is determined more by the conversion to protease resistance than by the initial binding step. These findings also imply that after the initial binding, further intermolecular interactions between PrP-sen and PrP-res are required to complete the process of conversion to the protease-resistant state.
Induction of natural competence in
Streptococcus pneumoniae
triggers lysis and DNA release from a subfraction of the cell population
Authors: Steinmoen Hilde , Knutsen Eivind , Håvarstein Leiv Sigve
Published: July 2002

Naturally competent bacteria have the ability to take up free DNA from the surrounding medium and incorporate this DNA into their genomes by homologous recombination. In naturally competent
Streptococcus pneumoniae
, and related streptococcal species from the mitis phylogenetic group, the competent state is not a constitutive property but is induced by a peptide pheromone through a quorum-sensing mechanism. Recent studies have shown that natural genetic transformation is an important mechanism for gene exchange between streptococci in nature. A prerequisite for effective gene exchange is the presence of streptococcal donor DNA in the environment. Despite decades of study of the transformation process we still do not know how this donor DNA is released from streptococcal cells to the external milieu. Traditionally, it has been assumed that donor DNA originates from cells that die and fall apart from natural causes. In this study we show that induction of the competent state initiates release of DNA from a subfraction of the bacterial population, probably by cell lysis. The majority of the cells induced to competence take up DNA and act as recipients, whereas the rest release DNA and act as donors. These findings show that natural transformation in streptococci provides a natural mechanism for genetic recombination that resembles sex in higher organisms.
A critical concentration of neutrophils is required for effective bacterial killing in suspension
Authors: Li Yongmei , Karlin Arthur , Loike John D. , Silverstein Samuel C.
Published: July 2002

We have examined the effect of neutrophil concentration on killing of a clinical isolate ofStaphylococcusepidermidis. Human neutrophils at concentrations varying from 105to 107per ml were mixed in suspension withS. epidermidisat concentrations varying from 103to 108colony-forming units/ml, and the concentration of viable bacteria was assayed after various times at 37°C. The rate of bacterial killing depended on the concentration of neutrophils and not on the ratio of neutrophils to bacteria. Below a critical concentration of neutrophils, bacteria growth was greater than neutrophil killing of bacteria even when the ratio of neutrophils to bacteria was 100:1. We fitted the time course of bacterial concentration and its dependence on neutrophil concentration with an exponential function, the exponent of which is (−kp+g)t, wherekis the second-order rate constant for bacterial killing,pis the neutrophil concentration,gis the first-order rate constant for bacterial growth, andtis time. We found thatk≈ 2 × 10−8ml per neutrophil per min, andg≈ 8 × 10−3/min. Only whenpis greater thang/k, which we call the critical neutrophil concentration, does the bacterial concentration fall. Under optimal assay conditions, the critical neutrophil concentration was 3–4 × 105per ml, a value very close to that (≤5 × 105per ml) known to predispose humans to bacterial and fungal infections.
Genetic drift and within-host metapopulation dynamics of HIV-1 infection
Authors: Frost Simon D. W. , Dumaurier Marie-Jeanne , Wain-Hobson Simon , Brown Andrew J. Leigh
Published: July 2002

Most HIV replication occurs in solid lymphoid tissue, which has prominent architecture at the histological level, which separates groups of productively infected CD4+cells. Nevertheless, current population models of HIV assume panmixis within lymphoid tissue. We present a simple “metapopulation” model of HIV replication, where the population of infected cells is comprised of a large number of small populations, each of which is established by a few founder viruses and undergoes turnover. To test this model, we analyzed viral genetic variation of infected cell subpopulations within the spleen and demonstrated the action of founder effects as well as significant variation in the extent of genetic differentiation between subpopulations among patients. The combination of founder effects and subpopulation turnover can result in an effective population size much lower than the actual population size and may contribute to the importance of genetic drift in HIV evolution despite a large number of infected cells.
Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells
Authors: Tanaka Hisashi , Tapscott Stephen J. , Trask Barbara J. , Yao Meng-Chao
Published: July 2002

Gene amplification is a common form of genomic instability in a wide variety of organisms and is often associated with tumor progression in mammals. One striking feature of many amplified genes is their organization as large inverted duplications (palindromes). Here, we describe a molecular mechanism for palindrome formation in mammalian cells that is also conserved in protists. We introduced a short (79 or 229 bp) inverted repeat into the genome of Chinese hamster ovary cells and showed that it promoted the formation of a large DNA palindrome after an adjacent DNA double-strand break. This finding suggests that short inverted repeats in the mammalian genome can have a critical role in the initiation of gene amplification. This specific mechanism may provide a novel target for cancer therapies.
In organello
formaldehyde crosslinking of proteins to mtDNA: Identification of bifunctional proteins
Authors: Kaufman Brett A. , Newman Scott M. , Hallberg Richard L. , Slaughter Clive A. , Perlman Philip S. , Butow Ronald A.
Published: July 2002

The segregating unit of mtDNA is a protein–DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an
in organello
formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected ≈20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that
hsp60-ts
mutants have a petite-inducing phenotype at the permissive temperature and that a
kgd2Δ
mutation increases the petite-inducing phenotype of an
abf2Δ
mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of ρ
+
mtDNA.
A male germ cell tumor-susceptibility-determining locus,
pgct1
, identified on murine chromosome 13
Authors: Muller Alexander J. , Teresky Angelika K. , Levine Arnold J.
Published: July 2002

Inbred 129 strain mice are predisposed to developing male germ cell tumors (GCTs) of the testes. The inherent genetic defects that underlie male GCT susceptibility in the 129 mouse strain are unknown. GCT incidence is increased in 129 strain males that lack functional p53 protein, and we have used this finding to facilitate the generation of panels of GCT-bearing intercross and backcross mice for genetic mapping analysis. A 129 strain locus, designated
pgct1
, that segregates with the male GCT phenotype has been identified on chromosome 13 near
D13Mit188
. This region of murine chromosome 13 may be syntenic to a portion of human chromosome 5q that is implicated in male GCT susceptibility in humans.
Biosynthesis of the thiazole moiety of thiamin in
Escherichia coli
: Identification of an acyldisulfide-linked protein–protein conjugate that is functionally analogous to the ubiquitin/E1 complex
Authors: Xi Jun , Ge Ying , Kinsland Cynthia , McLafferty Fred W. , Begley Tadhg P.
Published: July 2002

A covalently linked protein–protein conjugate between ThiF and ThiS thiocarboxylate was found in a partially purified coexpressed ThiF/ThiS protein mixture by using Fourier transform mass spectrometry. The Cys-184 of ThiF and the C terminus of ThiS thiocarboxylate were identified to be involved in the formation of this complex by using both mutagenesis and chemical modification methods. A complementation study of

Escherichia coli thiF
−

using
thiF
(C184S) suggests that this conjugate is an essential intermediate involved in the biosynthesis of the thiazole moiety of thiamin. This ThiF/ThiS conjugate is the first characterized example of a unique acyldisulfide intermediate in a biosynthetic system. This protein conjugate is also an example of an ubiquitin-E1 like protein–protein conjugate in prokaryotes and supports a strong evolutionary link between thiamin biosynthesis and the ubiquitin conjugating system.
Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways
Authors: Ying Ying , Qi Xiaoxia , Zhao Guang-Quan
Published: July 2002

Extraembryonic ectoderm-derived factors instruct the pluripotent epiblast cells to develop toward a restricted primordial germ cell (PGC) fate during murine gastrulation. Genes encoding
Bmp4
of the Dpp class and
Bmp8b
of the 60A class are expressed in the extraembryonic ectoderm and targeted mutation of either results in severe defects in PGC formation. It has been shown that heterodimers of DPP and 60A classes of bone morphogenetic proteins (BMPs) are more potent than each homodimers in bone and mesoderm induction
in vitro
, suggesting that BMP4 and BMP8B may form heterodimers to induce PGCs. To investigate how BMP4 and BMP8B interact and signal for PGC induction, we cocultured epiblasts of embryonic day 6.0–6.25 embryos with BMP4 and BMP8B proteins produced by COS cells. Our data show that BMP4 or BMP8B homodimers alone cannot induce PGCs whereas they can in combination, providing evidence that two BMP pathways are simultaneously required for the generation of a given cell type in mammals and also providing a prototype method for PGC induction
in vitro
. Furthermore, the PGC defects of
Bmp8b
mutants can be rescued by BMP8B homodimers whereas BMP4 homodimers cannot mitigate the PGC defects of
Bmp4
null mutants, suggesting that BMP4 proteins are also required for epiblast cells to gain germ-line competency before the synergistic action of BMP4 and BMP8B.
An essential role of a TatC homologue of a ΔpH- dependent protein transporter in thylakoid membrane formation during chloroplast development in
Arabidopsis thaliana
Authors: Motohashi Reiko , Nagata Noriko , Ito Takuya , Takahashi Seiji , Hobo Tokunori , Yoshida Shigeo , Shinozaki Kazuo
Published: July 2002

At least three transport systems function in targeting nuclear-encoded chloroplast proteins to the chloroplast thylakoid membrane. One of these systems requires a thylakoid pH gradient and is named the ΔpH-dependent protein transport system. A similar ΔpH export system of
Escherichia coli
contains four components, twin arginine translocation A (TatA), TatB, TatC, and TatE. TatC is a major component of the ΔpH-dependent protein transporter in
E. coli
and functions in the translocation of tightly folded proteins across membranes. We have isolated four transposon-inserted albino mutants named albino and pale green 2 (
apg2
) from
Arabidopsis thaliana
and showed that the transposons were inserted into different sites of a single gene. The
APG2
gene product (named cpTatC) has sequence similarity with bacterial TatC and contains six putative transmembrane domains, including bacterial TatC proteins and a transit peptide in its N terminus.
apg2
mutants showed albino phenotypes and could not grow in soil. The
apg2
plastids were highly vacuolated, lacked internal membrane structures and lamellae of the thylakoid membrane, and contained many densely stained globule structures, like undifferentiated proplastids. Immunoblot analysis detected no thylakoid membrane proteins such as D1, light-harvesting complex, and OE23 in
apg2
plastids, whereas soluble proteins such as rubisco large and small subunits were not decreased. These results indicate an essential role of cpTatC in chloroplast development, especially in thylakoid membrane formation.
Complete, long-lasting protection against malaria of mice primed and boosted with two distinct viral vectors expressing the same plasmodial antigen
Authors: Bruña-Romero Oscar , González-Aseguinolaza Gloria , Hafalla Julius C. R. , Tsuji Moriya , Nussenzweig Ruth S.
Published: July 2002

We report that complete protection against malaria and total inhibition of liver stage development and parasitemia was obtained in 100% of BALB/c mice primed with a replication-defective recombinant adenovirus expressing the circumsporozoite (CS) protein ofPlasmodium yoelii(AdPyCS), followed by a booster with an attenuated recombinant vaccinia virus, expressing the same malaria antigen, VacPyCS. We found increased levels of activated CS-specific CD8+and CD4+T cells, higher anti-sporozoite antibody titers, and greater protection in these mice, when the time between priming and boosting with these two viral vectors was extended from 2 to 8 or more weeks. Most importantly, by using this immunization regimen, the protection of the immunized mice was found to be long-lasting, namely complete resistance to infection of all animals 3 1/2 months after priming. These results indicate that immunization with AdPyCS generates highly effective memory T and B cells that can be recalled long after priming by boosting with VacPyCS.
Myxococcus xanthus
fibril appendages are essential for excitation by a phospholipid attractant
Authors: Kearns Daniel B. , Campbell Blakemore D. , Shimkets Lawrence J.
Published: July 2002

Isolated (A-motile)
Myxococcus xanthus
cells glide
over solid surfaces and display excitation, a suppression of direction
reversals, when presented with phosphatidylethanolamine (PE) purified
from its own membranes or synthetic dilauroyl PE and dioleoyl PE.
Although the mechanism of PE signal transduction is unknown, we
hypothesized that
M. xanthus
might use
surface-associated factors to detect exogenous PE to prevent
endogenous lipids from self-stimulating the sensory system.
Peritrichous protein and polysaccharide appendages called fibrils were
correlated with dilauroyl PE excitation. Wild-type cells treated with
Congo red, an inhibitor of fibril assembly, and mutants defective in
fibril biosynthesis showed an elevated reversal period, which suggested
that fibrils regulate the gliding motor. Furthermore, the loss of
fibrils resulted in loss of excitation to dilauroyl PE but not dioleoyl
PE. Restoration of fibril production to these mutants restored the
dilauroyl PE response. In addition, the
dif
cytoplasmic
signal transduction system and starvation conditions were required for
dilauroyl PE excitation. The chemically specific nature of the response
and the dependence on the
dif
system suggests that
fibrils define a novel sensory organelle whose evolution may have been
necessary to prevent autostimulation by endogenous membrane
lipids. Because the hydrophobic nature of dilauroyl PE would be
inaccessible to periplasmic chemosensors, we suggest that fibrils act
as extracellular signal transducers to probe surfaces for insoluble
chemical signals.
Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells
Authors: Kanovsky Mecheal , Raffo Anthony , Drew Lisa , Rosal Ramon , Do Tamara , Friedman Fred K. , Rubinstein Pablo , Visser Jan , Robinson Richard , Brandt-Rauf Paul W. , Michl Josef , Fine Robert L. , Pincus Matthew R.
Published: July 2002

We have synthesized three peptides from the mdm-2 binding domain of human p53, residues 12–26 (PPLSQETFSDLWKLL), residues 12–20, and 17–26. To enable transport of the peptides across the cell membrane and at the same time to maximize the active mdm-2 binding α-helical conformation for these peptides, each was attached at its carboxyl terminus to the penetratin sequence, KKWKMRRNQFWVKVQRG, that contains many positively charged residues that stabilize an α-helix when present on its carboxyl terminal end. All three peptides were cytotoxic to human cancer cells in culture, whereas a control, unrelated peptide attached to the same penetratin sequence had no effect on these cell lines. The same three cytotoxic peptides had no effect on the growth of normal cells, including human cord blood-derived stem cells. These peptides were as effective in causing cell death in p53-null cancer cells as in those having mutant or normal p53. Peptide-induced cell death is not accompanied by expression of apoptosis-associated proteins such as Bax and
waf
p21
. Based on these findings, we conclude that the antiproliferative effects of these p53-derived peptides are not completely dependent on p53 activity and may prove useful as general anticancer agents.
Effect of
Bradyrhizobium
photosynthesis on stem nodulation of
Aeschynomene sensitiva
Authors: Giraud Eric , Hannibal Laure , Fardoux Joel , Verméglio Andre , Dreyfus Bernard
Published: July 2002

Some leguminous species of the genus
Aeschynomene
are specifically stem-nodulated by photosynthetic bradyrhizobia. To
study the effect of bacterial photosynthesis during symbiosis, we
generated a photosynthesis-negative mutant of the
Bradyrhizobium
sp. strain ORS278 symbiont of
Aeschynomene sensitiva
. The presence of a functional
photosynthetic unit in bacteroids and the high expression of the
photosynthetic genes observed in stem nodules demonstrate that the
bacteria are photosynthetically active during stem symbiosis. Stem
inoculation by the photosynthetic mutant gave a 50% decrease in
stem-nodule number, which reduced nitrogen fixation activity and plant
growth in the same proportion. These results indicate an important role
of bacterial photosynthesis in the efficiency of stem nodulation.
The TRAP/Mediator coactivator complex interacts directly with estrogen receptors α and β through the TRAP220 subunit and directly enhances estrogen receptor function
in
vitro
Authors: Kang Yun Kyoung , Guermah Mohamed , Yuan Chao-Xing , Roeder Robert G.
Published: July 2002

Target gene activation by nuclear hormone receptors, including estrogen receptors (ERs), is thought to be mediated by a variety of interacting cofactors. Here we identify a number of nuclear extract-derived proteins that interact with immobilized ER ligand binding domains in a 17β-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)/Mediator coactivator complex, which interacts with ERα and ERβ in both unfractionated nuclear extracts and purified form. Studies with extracts from TRAP220
−/−
fibroblasts reveal that these interactions depend on TRAP220, a TRAP/Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the
in vitro
interaction is documented further by the isolation of an ERα–TRAP/Mediator complex from cultured cells expressing an epitope-tagged ERα. Finally, the complete TRAP/Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP/Mediator, through TRAP220, in ER function.
Mass vaccination to control chickenpox: the influence of zoster.
Authors: Ferguson N M , Anderson R M , Garnett G P
Published: July 2002

The impact of transmission events from patients with shingles (zoster) on the epidemiology of varicella is examined before and after the introduction of mass immunization by using a stochastic mathematical model of transmission dynamics. Reactivation of the virus is shown to damp stochastic fluctuations and move the dynamics toward simple annual oscillations. The force of infection due to zoster cases is estimated by comparison of simulated and observed incidence time series. The presence of infectious zoster cases reduces the tendency for mass immunization to increase varicella incidence at older ages when disease severity is typically greater.
Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.
Authors: Park M S , Knauf J A , Pendergrass S H , Coulon C H , Strniste G F , Marrone B L , MacInnes M A
Published: July 2002

Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. Using confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number of XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using beta-galactosidase-XPG fusion constructs (beta-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized beta-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic beta-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus.
Disruption of epithelial gamma delta T cell repertoires by mutation of the Syk tyrosine kinase.
Authors: Mallick-Wood C A , Pao W , Cheng A M , Lewis J M , Kulkarni S , Bolen J B , Rowley B , Tigelaar R E , Pawson T , Hayday A C
Published: July 2002

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.