bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of colitis by a CD4+T cell clone specific for a bacterial epitope
Authors: Kullberg Marika C. , Andersen John F. , Gorelick Peter L. , Caspar Patricia , Suerbaum Sebastian , Fox James G. , Cheever Allen W. , Jankovic Dragana , Sher Alan
Published: December 2003

It is now well established that the intestinal flora plays an important role in the pathogenesis of inflammatory bowel disease (IBD). However, whether bacteria serve as the sole target of the immune response in this process or whether they act indirectly by triggering an anti-self response is still unclear. We have previously shown that specific pathogen-free IL-10-deficient (IL-10 KO) mice develop a T helper (Th1)-cytokine associated colitis after experimental infection withHelicobacter hepaticus. We here show thatH. hepaticusAg (SHelAg)-specific CD4+Th1 clones transfer disease toH. hepaticus-infected T cell-deficient RAG KO hosts. Importantly, uninfected recipients of the SHelAg-specific clones did not develop intestinal inflammation, and a controlSchistosoma mansoni-specific Th1 clone did not induce colitis upon transfer to infected RAG KO mice. The disease-inducing T cell clones recognized antigen(s) (Ag) specifically expressed by certainHelicobacterspecies as they responded when stimulatedin vitrowithH. hepaticusandHelicobacter typhloniusAg, but not when cultured with Ag preparations fromHelicobacter pylori, various non-helicobacter bacteria, or with cecal bacterial lysate from uninfected mice. Characterization of the Ag specificity of one of the clones showed that it reacts uniquely with a 15-mer peptide epitope on the flagellar hook protein (FlgE) ofH. hepaticuspresented by I-Ab. Together, our results demonstrate that colitis can be induced by clonal T cell populations that are highly specific for target Ag on intestinal bacteria, suggesting that an aberrant T cell response directed against gut flora is sufficient to trigger IBD.
Binding ofPlasmodium falciparum-infected erythrocytes to the membrane-bound form of Fractalkine/CX3CL1
Authors: Hatabu Toshimitsu , Kawazu Shin-ichiro , Aikawa Masamichi , Kano Shigeyuki
Published: December 2003

Plasmodium falciparum-infected erythrocytes (pRBCs) adhere to the endothelium via receptors expressed on the surface of vascular endothelial cells (EC) and sequester in the microvasculature of several organs and block the blood circulation. The sequestration, which involves receptors, may be related to the severity of malaria. Here, we report that pRBCs bind to the membrane-bound form of Fractalkine/CX3CL1 (FKN), which is expressed on the surface of vascular EC in various organs. pRBCs adhered to FKN on the surface of FKN cDNA-transfected Chinese hamster ovary cells (CHO-FKN cells). Both the recombinant human FKN-chemokine domain (FKN-CD) and anti-FKN-CD antibody efficiently blocked adherence of pRBCs to CHO-FKN cells. Similar to binding between FKN and FKN receptor on blood mononuclear cells, two amino acid residues, Lys-7 and Arg-47 within FKN-CD, were critical for FKN-pRBC binding. Immunohistological analysis revealed the expression of FKN on EC at the site of sequestration in the brain of a patient with cerebral malaria. These results suggest that the membrane-bound form of FKN acts as a receptor for pRBCs, and this may contribute to furthering our present understanding of cytoadherence in the pathology of falciparum malaria.
Fundamental role of the Rip2/caspase-1 pathway in hypoxia and ischemia-induced neuronal cell death
Authors: Zhang Wen-Hua , Wang Xin , Narayanan Malini , Zhang Yu , Huo Chunfeng , Reed John C. , Friedlander Robert M.
Published: December 2003

Caspase-1 plays a key role in inflammatory pathways by processing pro-IL-1β into the active cytokine mature IL-1β. Given its sequence similarity with the
Caenorhabditis elegans
cell death gene
ced-3
,it has long been speculated that caspase-1 may also play a role in cell death. However, an unequivocal role for caspase-1 in cell death has been questioned, and not definitively demonstrated. Furthermore, if caspase-1 does play a role in cell death, its position in the apoptotic hierarchy has not been clearly defined. Previous studies have shown that caspase-1 knockout (KO) mice and transgenic mice expressing a dominant-negative caspase-1 construct are resistant to ischemic brain injury. We provide direct evidence that caspase-1 plays a key role in neuronal cell death and that caspase-1 is an apical activator of the cell death pathway in the premitochondrial collapse stage. Furthermore, we demonstrate that Rip2/Cardiak/Rick is a stress-inducible upstream modulator of pro-caspase-1 apoptotic activation. We provide evidence that Bid cleavage appears to be an important downstream effector of caspase-1-mediated cell death. Our data demonstrate that caspase-1 is an apical mediator of neuronal cell death during
in vitro
hypoxia, and confirmed
in vivo
in ischemia, and provide insights into the sequence of events involved in this pathological cell death process.
An efficient proteomics method to identify the cellular targets of protein kinase inhibitors
Authors: Godl Klaus , Wissing Josef , Kurtenbach Alexander , Habenberger Peter , Blencke Stephanie , Gutbrod Heidrun , Salassidis Kostadinos , Stein-Gerlach Matthias , Missio Andrea , Cotten Matt , Daub Henrik
Published: December 2003

Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580.
In vitro
kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38α whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.
Divergent tRNA-like element supports initiation, elongation, and termination of protein biosynthesis
Authors: Jan Eric , Kinzy Terri Goss , Sarnow Peter
Published: December 2003

The cricket paralysis virus internal ribosome entry site (IRES) can, in the absence of canonical initiation factors and initiator tRNA (Met-tRNA
i
), occupy the ribosomal P-site and assemble 80S ribosomes. Here we show that the IRES assembles mRNA-80S ribosome complexes by recruitment of 60S subunits to preformed IRES-40S complexes. Addition of eukaryotic elongation factors eEF1A and eEF2 and aminoacylated elongator tRNAs resulted in the synthesis of peptides, implying that the IRES RNA itself mimics the function of Met-tRNA
i
in the P-site to trigger the first translocation step without peptide bond formation. IRES-80S complexes that contained a stop codon in the A-site recruited eukaryotic release factor eRF1, resulting in ribosome rearrangements in a surprisingly eEF2-dependent manner. Thus, this P-site-occupying IRES directs the assembly of 80S ribosomes, sets the translational reading frame, and mimics the functions of both Met-tRNA
i
and peptidyl tRNA to support elongation and termination.
Controlling system dimension: A class of real systems that obey the Kaplan–Yorke conjecture
Authors: Nichols J. M. , Todd M. D. , Seaver M. , Trickey S. T. , Pecora L. M. , Moniz L.
Published: December 2003

The Kaplan–Yorke conjecture suggests a simple relationship between the fractal dimension of a system and its Lyapunov spectrum. This relationship has important consequences in the broad field of nonlinear dynamics where dimension and Lyapunov exponents are frequently used descriptors of system dynamics. We develop an experimental system with controllable dimension by making use of the Kaplan–Yorke conjecture. A rectangular steel plate is driven with the output of a chaotic oscillator. We controlled the Lyapunov exponents of the driving and then computed the fractal dimension of the plate's response. The Kaplan–Yorke relationship predicted the system's dimension extremely well. This finding strongly suggests that other driven linear systems will behave similarly. The ability to control the dimension of a structure's vibrational response is important in the field of vibration-based structural health monitoring for the robust extraction of damage-sensitive features.
Fungal farming in a snail
Authors: Silliman Brian R. , Newell Steven Y.
Published: December 2003

Mutualisms between fungi and fungus-growing animals are model systems for studying coevolution and complex interactions between species. Fungal growing behavior has enabled cultivating animals to rise to major ecological importance, but evolution of farming symbioses is thought to be restricted to three terrestrial insect lineages. Surveys along 2,000 km of North America's Atlantic coast documented that the marine snail
Littoraria irrorata
grazes fungus-infected wounds on live marsh grass throughout its range. Field experiments demonstrate a facultative, farming mutualism between
Littoraria
and intertidal fungi. Snails graze live grass primarily not to feed but to prepare substrate for fungal growth and consume invasive fungi. Fungal removal experiments show that snails and fungi act synergistically to suppress marsh grass production. These results provide a case of fungus farming in the marine environment and outside the class Insecta and reveal a previously undemonstrated ecological mechanism (i.e., facilitation of fungal invasion) by which grazers can exert top-down control of marine plant production.
Contribution of transient receptor potential channels to the control of GABA release from dendrites
Authors: Munsch Thomas , Freichel Marc , Flockerzi Veit , Pape Hans-Christian
Published: December 2003

Neuronal dendrites have been shown to actively contribute to synaptic information transfer through the Ca
2+
-dependent release of neurotransmitter, although the underlying mechanisms remain elusive. This study shows that the increase in dendritic γ-aminobutyric acid (GABA) release from thalamic interneurons mediated by the activation of 5-hydroxytryptamine type 2 receptors requires Ca
2+
entry that does not involve Ca
2+
release nor voltage-gated Ca
2+
channels in the plasma membrane but that is critically dependent on the transient receptor potential (TRP) protein TRPC4. These data ascribe a functional role of agonist-activated TRP channels to the release of transmitters from dendrites, thereby indicating a principle underlying synaptic interactions in the brain.
Dynamic analysis of larval locomotion in
Drosophila
chordotonal organ mutants
Authors: Caldwell Jason C. , Miller Matthew M. , Wing Susan , Soll David R. , Eberl Daniel F.
Published: December 2003

Rhythmic movements, such as peristaltic contraction, are initiated by output from central pattern generator (CPG) networks in the CNS. These oscillatory networks elicit locomotion in the absence of external sensory or descending inputs, but CPG circuits produce more directed and behaviorally relevant movement via peripheral nervous system (PNS) input.
Drosophila melanogaster
larval locomotion results from patterned muscle contractions moving stereotypically along the body segments, but without PNS feedback, contraction of body segments is uncoordinated. We have dissected the role of a subset of mechanosensory neurons in the larval PNS, the chordotonal organs (chos), in providing sensory feedback to the locomotor CPG circuit with
dias
(Dynamic Image Analysis System) software. We analyzed mutants carrying cho mutations including
atonal
, a cho proneural gene,
beethoven
, a cho cilia class mutant,
smetana
and
touch-insensitive larva B
, two axonemal mutants, and
5D10
, a weak cho mutant. All cho mutants have defects in gross path morphology compared to controls. These mutants exhibit increased frequency and duration of turning (decision-making) and reduced duration of linear locomotion. Furthermore, cho mutants affect locomotor parameters, including reduced average speed, direction change, and persistence.
dias
analysis of peristaltic waves indicates that mutants exhibit reduced average speed, positive flow and negative flow, and increased stride period. Thus, cho sensilla are major proprioceptive components that underlie touch sensitivity, locomotion, and peristaltic contraction by providing sensory feedback to the locomotor CPG circuit in larvae.
Divergence of
Hoxc8
early enhancer parallels diverged axial morphologies between mammals and fishes
Authors: Anand Sanjay , Wang Wayne C. H. , Powell Dennis R. , Bolanowski Stacey A. , Zhang Jian , Ledje Christina , Pawashe Aruna B. , Amemiya Chris T. , Shashikant Cooduvalli S.
Published: December 2003

There is considerable interest in understanding how cis-regulatory modifications drive morphological changes across species. Because developmental regulatory genes, including
Hox
genes, are remarkably conserved, their noncoding regulatory regions are likely sources for variations. Modifications of
Hox
cis-regulatory elements have potential to alter
Hox
gene expression and, hence, axial morphologies. In vertebrates, differences in the axial levels of
Hox
gene expression correlate with differences in the number and relative position of thoracic vertebrae. Variation in cis-regulatory elements of
Hox
genes can be identified by comparative sequence and reporter gene analyses in transgenic mouse embryos. Using these approaches, we show a remarkable divergence of the
Hoxc8
early enhancers between mammals and fishes representing diverse axial morphologies. Extensive restructuring of the
Hoxc8
early enhancer including nucleotide substitutions, inversion, and divergence result in distinct patterns of reporter gene expression along the embryonic axis. Our results provide an evolutionary perspective on how the enhancer elements are engineered and support the hypothesis that remodeling of
Hox
regulatory elements in different species has played a significant role in generating morphological diversity.
Glucose transporter-1-deficient mice exhibit impaired development and deformities that are similar to diabetic embryopathy
Authors: Heilig Charles W. , Saunders Thomas , Brosius Frank C. , Moley Kelle , Heilig Kathleen , Baggs Raymond , Guo LiRong , Conner David
Published: December 2003

The hyperglycemia of maternal diabetes suppresses the glucose transporter-1 (GLUT1) facilitative glucose transporter 49–66% in preimplantation embryos. Glucose uptake is reduced and apoptosis is activated. We hypothesized that the reduction of embryonic GLUT1 may play a key role in the malformations of diabetic embryopathy. Therefore, we produced GLUT1-deficient transgenic mice [i.e., antisense-GLUT1 (GT1AS)] to determine whether GLUT1 deficiency alone could reproduce the growth defects. Early cell division of fertilized mouse eggs injected with GT1AS was markedly impaired,
P
< 0.001 vs. controls. Two populations of preimplantation embryos obtained from GT1AS × GT1AS heterozygote matings exhibited reduction of the 2-deoxyglucose uptake rate: one by 50% (presumed heterozygotes,
P
< 0.001 vs. control) and the other by 95% (presumed homozygotes,
P
< 0.001 vs. heterozygotes). Embryonic GLUT1 deficiency in the range reported with maternal diabetes was associated with growth retardation and developmental malformations similar to those described in diabetes-exposed embryos: intrauterine growth retardation (31.1%), caudal regression (9.8%), anencephaly with absence of the head (6.6%), microphthalmia (4.9%), and micrognathia (1.6%). Reduced body weight (small embryos, <70% of the nontransgenic body weight) was accompanied by other malformations and a 56% reduction of GLUT1 protein,
P
< 0.001 vs. nonsmall embryos (body weight ≥70% normal). The heart, brain, and kidneys of embryonic day 18.5 GT1AS embryos exhibited 24–51% reductions of GLUT1 protein. The homozygous GT1AS genotype was lethal during gestation. Reduced embryonic GLUT1 was associated with the appearance of apoptosis. Therefore, GLUT1 deficiency may play a role in producing embryonic malformations resulting from the hyperglycemia of maternal diabetes. Late gestational macrosomia was absent, apparently requiring a different mechanism.
Circadian gene expression in mammalian fibroblasts revealed by real-time luminescence reporting: Temperature compensation and damping
Authors: Izumo Mariko , Johnson Carl Hirschie , Yamazaki Shin
Published: December 2003

Mammalian cells such as rat-1 fibroblasts have been shown to exhibit daily oscillations in the expression of several gene transcripts in culture. After induction, these oscillations persist with a period of ≈24 h for several days. This characteristic suggests that the oscillations are controlled by a circadian clock, but the crucial criterion of temperature compensation has not been demonstrated for rat-1 fibroblasts. We have developed an automated assay of circadian expression of the
mPer1
promoter in rat-1 fibroblasts that have been stably transfected with a luciferase reporter. Using this cell culture-based
in vitro
luminescent reporter assay, we found that the daily oscillation of
mPer1
promoter activity in rat-1 cells is temperature compensated over the range of 28.5-36.5°C. This finding means that these oscillations are bona fide circadian rhythms. Moreover, the circadian clock of these homeothermic mammalian cells not only is temperature compensated but also is overcompensated such that it runs faster at cooler temperatures (Q
10
of 0.85-0.88). The oscillations in rat-1 fibroblasts damp more rapidly at cooler temperatures, and damping is not due to cells becoming unhealthy because a second stimulus will reinitiate a robust rhythm. These data show that rat-1 cell cultures that are stably transfected with luminescence reporters are an excellent model system for studying circadian clocks at the cellular level in mammals.
Prediction and statistics of pseudoknots in RNA structures using exactly clustered stochastic simulations
Authors: Xayaphoummine A. , Bucher T. , Thalmann F. , Isambert H.
Published: December 2003

Ab initio
RNA secondary structure predictions have long dismissed helices interior to loops, so-called pseudoknots, despite their structural importance. Here we report that many pseudoknots can be predicted through long-time-scale RNA-folding simulations, which follow the stochastic closing and opening of individual RNA helices. The numerical efficacy of these stochastic simulations relies on an 𝒪(
n
2
) clustering algorithm that computes time averages over a continuously updated set of
n
reference structures. Applying this exact stochastic clustering approach, we typically obtain a 5- to 100-fold simulation speed-up for RNA sequences up to 400 bases, while the effective acceleration can be as high as 10
5
-fold for short, multistable molecules (≤150 bases). We performed extensive folding statistics on random and natural RNA sequences and found that pseudoknots are distributed unevenly among RNA structures and account for up to 30% of base pairs in G+C-rich RNA sequences (online RNA-folding kinetics server including pseudoknots:
http://kinefold.u-strasbg.fr
).
TGF-β-induced RhoA and p160
ROCK
activation is involved in the inhibition of Cdc25A with resultant cell-cycle arrest
Authors: Bhowmick Neil A. , Ghiassi Mayshan , Aakre Mary , Brown Kimberly , Singh Vikas , Moses Harold L.
Published: December 2003

The ability of the transforming growth factor β (TGF-β) signaling pathways to inhibit proliferation of most cells while stimulating proliferation of others remains a conundrum. In this article, we report that the absence of RhoA and p160
ROCK
activity in fibroblastic NIH 3T3 cells and its presence in epithelial NMuMG cells can at least partially explain the difference in the TGF-β growth response. Further, evidence is presented for TGF-β-stimulated p160
ROCK
translocation to the nucleus and inhibitory phosphorylation of the cyclin-dependent kinase-activating phosphatase, Cdc25A. The resultant suppression of Cdk2 activity contributes to G
1
/S inhibition in NMuMG cells. These data provide evidence that signaling through RhoA and p160
ROCK
is important in TGF-β inhibition of cell proliferation and links signaling components for epithelial transdifferentiation with regulation of cell-cycle progression.
Inhibition of mutant p53 expression and growth of DMS-153 small cell lung carcinoma by antagonists of growth hormone-releasing hormone and bombesin
Authors: Kanashiro Celia A. , Schally Andrew V. , Groot Kate , Armatis Patricia , Bernardino Andrea L. F. , Varga Jozsef L.
Published: December 2003

We investigated the effects of growth hormone-releasing hormone (GHRH) antagonists, JV-1-65 and JV-1-63, and bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC-3940-II on DMS-153 human small cell lung carcinoma xenografted into nude mice. Treatment with 10 μg/day JV-1-65 or RC-3940-II decreased tumor volume by 28% (
P
< 0.05) and 77% (
P
< 0.01), respectively, after 42 days compared with controls. Combination of JV-1-65 and RC-3940-II induced the greatest inhibition of tumor proliferation (95%;
P
< 0.01), suggesting a synergism. Western blotting showed that the antitumor effects of these antagonists were associated with inhibition of the expression of the mutant tumor suppressor protein p53 (Tp53). Mutation was detected by sequence analysis of the p53 gene at codon 155: ACC [Thr] → CCC [Pro]. Combination of JV-1-65 and RC-3940-II decreased the levels of mutant p53 protein by 42% (
P
< 0.01) compared with controls. JV-1-65, JV-1-63, and RC-3940-II, given singly, reduced mutant p53 protein expression by 18-24% (
P
< 0.05). Serum insulin-like growth factor (IGF)-I levels were diminished in animals receiving GHRH antagonists. mRNA levels for IGF-II, IGF receptor-I, GRP receptor, and EGF receptor in tumors were significantly decreased by combined treatment with JV-1-65 and RC-3940-II. DMS-153 tumors expressed mRNAs for GHRH and GHRH receptor splice variants 1 and 2, suggesting that GHRH could be an autocrine growth factor. Proliferation of DMS-153 cells
in vitro
was stimulated by GRP and IGF-II and inhibited by JV-1-65. This study indicates that GHRH antagonists and BN/GRP antagonist inhibit the growth of DMS-153 small cell lung carcinoma concomitantly with the expression of mutant Tp53, which might uncouple the signal transduction pathways for cell growth stimulation.
Genetic, biochemical, and morphological evidence for the involvement of N-glycosylation in biosynthesis of the cell wall β1,6-glucan ofSaccharomyces cerevisiae
Authors: Chavan Manasi , Suzuki Tadashi , Rekowicz Magdalena , Lennarz William
Published: December 2003

Recent evidence indicates that Stt3p plays a central role in the recognition and/or catalytic step in N-glycosylation (asparagine-linked glycosylation) in the lumen of the endoplasmic reticulum. It is known thatstt3mutants exhibit certain phenotypic features that are suggestive of a cell wall defect. To understand the basis of these phenotypes, we devised a genetic screen to isolate strains bearing mutations that lead to synthetic lethality in combination with thestt3–1mutation. Using this screen, we were surprised to identify twoKREgenes (KRE5andKRE9) that are involved in the biosynthesis of the cell wall β1,6-glucan. This finding led us to propose that the N-glycosylation process is essential in the biosynthesis of cell wall β1,6-glucan. This proposal was supported by the observation that severalstt3mutants exhibited a 60–70% reduction in the content of cell wall β1,6-glucan as compared with WT cells. Transmission electron microscopy revealed that thestt3mutant strains exhibit a diffused cell wall with loss of the outer mannoprotein layer as compared with the WT cells. Thus, we provide genetic, morphological, and biochemical evidence for the critical involvement of N-glycosylation in some step in assembly of the cell wall β1,6-glucan inSaccharomyces cerevisiae.
A role for mechanosensitive channels in survival of stationary phase: Regulation of channel expression by RpoS
Authors: Stokes Neil R. , Murray Heath D. , Subramaniam Chandrasekaran , Gourse Richard L. , Louis Petra , Bartlett Wendy , Miller Samantha , Booth Ian R.
Published: December 2003

The mechanosensitive (MS) channels MscS and MscL are essential for the survival of hypoosmotic shock by
Escherichia coli
cells. We demonstrate that MscS and MscL are induced by osmotic stress and by entry into stationary phase. Reduced levels of MS proteins and reduced expression of
mscL–
and
mscS
–
LacZ
fusions in an
rpoS
mutant strain suggested that the RNA polymerase holoenzyme containing σ
S
is responsible, at least in part, for regulating production of MS channel proteins. Consistent with the model that the effect of σ
S
is direct, the MscS and MscL promoters both use RNA polymerase containing σ
S
in vitro
. Conversely,
clpP
or
rssB
mutations, which cause enhanced levels of σ
S
, show increased MS channel protein synthesis. RpoS null mutants are sensitive to hypoosmotic shock upon entry into stationary phase. These data suggest that MscS and MscL are components of the RpoS regulon and play an important role in ensuring structural integrity in stationary phase bacteria.
A bacterial group II intron favors retrotransposition into plasmid targets
Authors: Ichiyanagi Kenji , Beauregard Arthur , Belfort Marlene
Published: December 2003

Group II introns, widely believed to be the ancestors of nuclear pre-mRNA introns, are catalytic RNAs found in bacteria, archaea, and eukaryotes. They are mobile genetic elements that move via an RNA intermediate. They retrohome to intronless alleles and retrotranspose to ectopic sites, aided by an intron-encoded protein with reverse transcriptase, maturase, and endonuclease activities. Many group II introns identified in bacteria reside on plasmid genomes rather than bacterial chromosomes, implying that plasmids are havens for these retroelements. This study demonstrates that almost one-fourth of retrotransposition events of the Ll.LtrB intron inLactococcus lactisare into the plasmid donor. This level is more than twice that predicted based on target size and plasmid copy number relative to the chromosome. In particular, the fraction of such plasmid targeting events was elevated to more than one-third of retrotransposition events by mutation of the intron-encoded endonuclease, a situation that may resemble most bacterial group II introns, which lack the endonuclease. Target-site sequences on the plasmid are more relaxed than those on the chromosome, likely accounting for preferred integration into plasmid replicons. Furthermore, the direction of integration relative to promoters and origins of replication is consistent with group II intron retrotransposition into single-stranded DNA at replication forks. This work provides mechanistic rationales for the prevalence of group II introns in natural plasmid populations and underscores that targeting to plasmids, which are themselves mobile elements, could promote intron spread.
Encephalization and allometric trajectories in the genusHomo: Evidence from the Neandertal and modern lineages
Authors: Bruner Emiliano , Manzi Giorgio , Arsuaga Juan Luis
Published: December 2003

The term “encephalization” is commonly used to describe an enlargement in brain size, considered as either absolute endocranial volumes or relative values in relation to body size. It is widely recognized that a considerable endocranial expansion occurred throughout the evolution of the genusHomo. This article aims to evaluate whether this phenomenon was the outcome of distinct evolutionary lineages, reaching similar brain expansions but through different trajectories. Endocranial morphology was studied in a sample of fossil hominines by multivariate approaches using both traditional metrics and geometric morphometrics. The analysis was focused on the transition from a generalized archaic pattern within the genusHomoto the modern morphology and compared with changes that occurred along the Neandertal lineage. The main result was the identification of two different evolutionary trajectories, in which a similar expansion in endocranial size has been reached by different changes in shape. Along the Neandertal lineage we observed maintenance of an “archaic” endocranial model, in which a large amount of variability is based on a single allometric trend. By contrast, when modern endocasts were compared with nonmodern ones, we found important differences apparently led by a parietal expansion. In this light, the origin of our species may have represented the opportunity to surpass the constraints imposed on encephalization by the ontogenetic pattern shared by nonmodernHomorepresentatives.
The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells
Authors: Chennathukuzhi Vargheese , Morales Carlos R. , El-Alfy Mohamed , Hecht Norman B.
Published: December 2003

Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of the human protein Translin, is a widely expressed and highly conserved protein with proposed functions in chromosomal translocations, mitotic cell division, and mRNA transport, stabilization, and storage. Targeted inactivation of TB-RBP leads to abnormalities in fertility and behavior. A testis-enriched kinesin KIF17b coimmunoprecipitates with TB-RBP in a RNA–protein complex containing specific cAMP-responsive element modulator (CREM)-regulated mRNAs. The specificity of this interaction is confirmed by
in vivo
RNA–protein crosslinking and transfections of hippocampal neurons. Combining
in situ
hybridization and immunohistochemistry at the electron microscope level, a temporally sequential dissociation of KIF17b and TB-RBP from specific mRNAs is detected with TB-RBP release coincident with the time of mRNA translation, indicating a separation of the processes of transport and translation. We conclude that KIF17b serves as a molecular motor component of a TB-RBP–mouse ribonucleoprotein complex transporting a group of specific CREM-regulated mRNAs in mammalian male postmeiotic germ cells. Because KIF17b has been reported to control CREM-dependent transcription in male germ cells by regulating the intracellular location of the transcriptional coactivator activator of CREM in testis, this indicates that one kinesin links the processes of transcription and transport of specific mRNAs in mammalian male germ cells.
Widespread purifying selection at polymorphic sites in human protein-coding loci
Authors: Hughes Austin L. , Packer Bernice , Welch Robert , Bergen Andrew W. , Chanock Stephen J. , Yeager Meredith
Published: December 2003

Estimation of gene diversity (heterozygosity) at 1,442 single-nucleotide polymorphism (SNP) loci in an ethnically diverse sample of humans revealed consistently reduced gene diversities at SNP loci causing amino acid changes, particularly those causing amino acid changes predicted to be disruptive to protein structure. The reduction of gene diversity at these SNP loci, in comparison to SNPs in the same genes not affecting protein structure, is evidence that negative natural selection (purifying selection) has reduced the population frequencies of deleterious SNP alleles. This, in turn, suggests that slightly deleterious mutations are widespread in the human population and that estimation of gene diversity even in a sample of modest size can help guide the search for disease-associated genes.
Gi protein activation in intact cells involves subunit rearrangement rather than dissociation
Authors: Bünemann Moritz , Frank Monika , Lohse Martin J.
Published: December 2003

G protein-coupled receptors transduce diverse extracellular signals, such as neurotransmitters, hormones, chemokines, and sensory stimuli, into intracellular responses through activation of heterotrimeric G proteins. G proteins play critical roles in determining specificity and kinetics of subsequent biological responses by modulation of effector proteins. We have developed a fluorescence resonance energy transfer (FRET)-based assay to directly measure mammalian G protein activation in intact cells and found that Gi proteins activate within 1-2 s, which is considerably slower than activation kinetics of the receptors themselves. More importantly, FRET measurements demonstrated that Gαi- and Gβγ-subunits do not dissociate during activation, as has been previously postulated. Based on FRET measurements between Gαi-yellow fluorescent protein and Gβγ-subunits that were fused to cyan fluorescent protein at various positions, we conclude that, instead, G protein subunits undergo a molecular rearrangement during activation. The detection of a persistent heterotrimeric composition during G protein activation will impact the understanding of how G proteins achieve subtype-selective coupling to effectors. This finding will be of particular interest for unraveling Gβγ-induced signaling pathways.
Homeoprotein DLX-1 interacts with Smad4 and blocks a signaling pathway from activin A in hematopoietic cells
Authors: Chiba Shigeru , Takeshita Kenichi , Imai Yoichi , Kumano Keiki , Kurokawa Mineo , Masuda Shigeo , Shimizu Kiyoshi , Nakamura Shuji , Ruddle Frank H. , Hirai Hisamaru
Published: December 2003

In the transforming growth factor β (TGF-β) superfamily, activin A, TGF-β1, and bone morphogenic protein 4 (BMP-4) have various effects on hematopoiesis, including early mesodermo-hematogenesis. After these cytokines bind to their respective receptor, a regulatory Smad is phosphorylated and becomes associated with Smad4, the common Smad, and the resulting complex translocates to the nucleus to regulate transcription. DLX1 is the product of a member of thedistal-lesshomeobox gene family, which is known to have important roles in embryogenesis, particularly in craniofacial development, and in GABAergic neurogenesis. DLX1 has been reported to be temporally and spatially coexpressed with BMP-4 during embryogenesis in selected contexts. We report here that, in addition to the previously reported regions/cells, DLX1 is expressed in hematopoietic cells in a lineage-dependent manner and that DLX1 interacts with Smad4 through its homeodomain. We show that it blocks multiple signals from TGF-β superfamily cytokines such as activin A, TGF-β1, and BMP-4, including differentiation of a hematopoietic cell line by activin A. Taken together, these data suggest that DLX1 may function as a regulator of multiple signals from TGF-β superfamily members in broad biological contexts during blood production.
Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation
Authors: Li Fengfu , Carlsson David , Lohmann Chris , Suuronen Erik , Vascotto Sandy , Kobuch Karin , Sheardown Heather , Munger Rejean , Nakamura Masatsugu , Griffith May
Published: December 2003

Our objective was to determine whether key properties of extracellular matrix (ECM) macromolecules can be replicated within tissue-engineered biosynthetic matrices to influence cellular properties and behavior. To achieve this, hydrated collagen and
N
-isopropylacrylamide copolymer-based ECMs were fabricated and tested on a corneal model. The structural and immunological simplicity of the cornea and importance of its extensive innervation for optimal functioning makes it an ideal test model. In addition, corneal failure is a clinically significant problem. Matrices were therefore designed to have the optical clarity and the proper dimensions, curvature, and biomechanical properties for use as corneal tissue replacements in transplantation.
In vitro
studies demonstrated that grafting of the laminin adhesion pentapeptide motif, YIGSR, to the hydrogels promoted epithelial stratification and neurite in-growth. Implants into pigs' corneas demonstrated successful
in vivo
regeneration of host corneal epithelium, stroma, and nerves. In particular, functional nerves were observed to rapidly regenerate in implants. By comparison, nerve regeneration in allograft controls was too slow to be observed during the experimental period, consistent with the behavior of human cornea transplants. Other corneal substitutes have been produced and tested, but here we report an implantable matrix that performs as a physiologically functional tissue substitute and not simply as a prosthetic device. These biosynthetic ECM replacements should have applicability to many areas of tissue engineering and regenerative medicine, especially where nerve function is required.
Adipose-specific peroxisome proliferator-activated receptor γ knockout causes insulin resistance in fat and liver but not in muscle
Authors: He Weimin , Barak Yaacov , Hevener Andrea , Olson Peter , Liao Debbie , Le Jamie , Nelson Michael , Ong Estelita , Olefsky Jerrold M. , Evans Ronald M.
Published: December 2003

Syndrome X, typified by obesity, insulin resistance (IR), dyslipidemia, and other metabolic abnormalities, is responsive to antidiabetic thiazolidinediones (TZDs). Peroxisome proliferator-activated receptor (PPAR) γ, a target of TZDs, is expressed abundantly in adipocytes, suggesting an important role for this tissue in the etiology and treatment of IR. Targeted deletion of PPARγ in adipose tissue resulted in marked adipocyte hypocellularity and hypertrophy, elevated levels of plasma free fatty acids and triglyceride, and decreased levels of plasma leptin and ACRP30. In addition, increased hepatic glucogenesis and IR were observed. Despite these defects, blood glucose, glucose and insulin tolerance, and insulin-stimulated muscle glucose uptake were all comparable to those of control mice. However, targeted mice were significantly more susceptible to high-fat diet-induced steatosis, hyperinsulinemia, and IR. Surprisingly, TZD treatment effectively reversed liver IR, whereas it failed to lower plasma free fatty acids. These results suggest that syndrome X may be comprised of separable PPARγ-dependent components whose origins and therapeutic sites may reside in distinct tissues.
Two different Swi5-containing protein complexes are involved in mating-type switching and recombination repair in fission yeast
Authors: Akamatsu Yufuko , Dziadkowiec Dorota , Ikeguchi Mitsunori , Shinagawa Hideo , Iwasaki Hiroshi
Published: December 2003

Homologous recombination is an important biological process that occurs in all organisms and facilitates genome rearrangements and repair of DNA double-strand breaks. Eukaryotic Rad51 proteins (Rad51
sp
or Rhp51 in fission yeast) are functional and structural homologs of bacterial RecA protein, an evolutionarily conserved protein that plays a key role in homologous pairing and strand exchange between homologous DNA molecules
in vitro
. Here we show that the fission yeast
swi5
+
gene, which was originally identified as a gene required for normal mating-type switching, encodes a protein conserved among eukaryotes and is involved in a previously uncharacterized Rhp51 (Rad51
sp
)-dependent recombination repair pathway that does not require the Rhp55/57 (Rad55/57
sp
) function. Protein interactions with both Swi5 and Rhp51 were found to be mediated by a domain common to Swi2 and Sfr1 (
S
wi
f
ive-dependent
r
ecombination repair protein
1
, a previously uncharacterized protein with sequence similarity to the C-terminal part of Swi2). Genetic epistasis analyses suggest that the Swi5–Sfr1–Rhp51 interactions function specifically in DNA recombination repair, whereas the Swi5–Swi2–Rhp51 interactions may function, together with chromodomain protein Swi6 (HP1 homolog), in mating-type switching.
A macroscopic scale model of bacterial flagellar bundling
Authors: Kim MunJu , Bird James C. , Van Parys Annemarie J. , Breuer Kenneth S. , Powers Thomas R.
Published: December 2003

Escherichia coli
and other bacteria use rotating helical filaments to swim. Each cell typically has about four filaments, which bundle or disperse depending on the sense of motor rotation. To study the bundling process, we built a macroscopic scale model consisting of stepper motor-driven polymer helices in a tank filled with a high-viscosity silicone oil. The Reynolds number, the ratio of viscous to elastic stresses, and the helix geometry of our experimental model approximately match the corresponding quantities of the full-scale
E. coli
cells. We analyze digital video images of the rotating helices to show that the initial rate of bundling is proportional to the motor frequency and is independent of the characteristic relaxation time of the filament. We also determine which combinations of helix handedness and sense of motor rotation lead to bundling.
Polariton lasing vs. photon lasing in a semiconductor microcavity
Authors: Deng Hui , Weihs Gregor , Snoke David , Bloch Jacqueline , Yamamoto Yoshihisa
Published: December 2003

Nearly one decade after the first observation of Bose–Einstein condensation in atom vapors and realization of matter-wave (atom) lasers, similar concepts have been demonstrated recently for polaritons: half-matter, half-light quasiparticles in semiconductor microcavities. The half-light nature of polaritons makes polariton lasers promising as a new source of coherent and nonclassical light with extremely low threshold energy. The half-matter nature makes polariton lasers a unique test bed for many-body theories and cavity quantum electrodynamics. In this article, we present a series of experimental studies of a polariton laser, exploring its properties as a relatively dense degenerate Bose gas and comparing it to a photon laser achieved in the same structure. The polaritons have an effective mass that is twice the cavity photon effective mass, yet seven orders of magnitude less than the hydrogen atom mass; hence, they can potentially condense at temperatures seven orders of magnitude higher than those required for atom Bose–Einstein condensations. Accompanying the phase transition, a polariton laser emits coherent light but at a threshold carrier density two orders of magnitude lower than that needed for a normal photon laser in a same structure. It also is shown that, beyond threshold, the polariton population splits to a thermal equilibrium Bose–Einstein distribution at in-plane wave number
k
∥
> 0 and a nonequilibrium condensate at
k
∥
> 0, with a chemical potential approaching to zero. The spatial distributions and polarization characteristics of polaritons also are discussed as unique signatures of a polariton laser.
Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium
Authors: Coakley Raymond D. , Grubb Barbara R. , Paradiso Anthony M. , Gatzy John T. , Johnson Larry G. , Kreda Sylvia M. , O'Neal Wanda K. , Boucher Richard C.
Published: December 2003

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-dependent airway epithelial bicarbonate transport is hypothesized to participate in airway surface liquid pH regulation and contribute to lung defense. We measured pH and ionic composition in apical surface liquid (ASL) on polarized normal (NL) and CF primary bronchial epithelial cell cultures under basal conditions, after cAMP stimulation, and after challenge with luminal acid loads. Under basal conditions, CF epithelia acidified ASL more rapidly than NL epithelia. Two ASL pH regulatory paths that contributed to basal pH were identified in the apical membrane of airway epithelia, and their activities were measured. We detected a ouabain-sensitive (nongastric) H+,K+-ATPase that acidified ASL, but its activity was not different in NL and CF cultures. We also detected the following evidence for a CFTR-dependent\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}secretory pathway that was defective in CF: (i) ASL [\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}] was higher in NL than CF ASL; (ii) activating CFTR with forskolin/3-isobutyl-1-methylxanthine alkalinized NL ASL but acidified CF ASL; and (iii) NL airway epithelia more rapidly and effectively alkalinized ASL in response to a luminal acid challenge than CF epithelia. We conclude that cultured human CF bronchial epithelial pHASLis abnormally regulated under basal conditions because of absent CFTR-dependent\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document}secretion and that this defect can lead to an impaired capacity to respond to airway conditions associated with acidification of ASL.
Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions
Authors: Zhu Ping , Chertova Elena , Bess Julian , Lifson Jeffrey D. , Arthur Larry O. , Liu Jun , Taylor Kenneth A. , Roux Kenneth H.
Published: December 2003

We used electron tomography to directly visualize trilobed presumptive envelope (env) glycoprotein structures on the surface of negatively stained HIV type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions. Wild–type HIV-1 and SIV virions had an average of 8–10 trimers per virion, consistent with predictions based on biochemical evidence. Mutant SIVs, biochemically demonstrated to contain high levels of the viral env proteins, averaged 70–79 trimers per virion in tomograms. These correlations strongly indicate that the visualized trimers represent env spikes. The env trimers were without obvious geometric distribution pattern or preferred rotational orientation. Combined with biochemical analysis of gag/env ratios in virions, these trimer counts allow calculation of the number of gag molecules per virion, yielding an average value of ≈1,400. Virion and env dimensions were also determined. Image-averaging analysis of SIV env trimers revealed a distinct chirality and strong concordance with recent molecular models. The results directly demonstrate the presence of env trimers on the surface of AIDS virus virions, albeit at numbers much lower than generally appreciated, and have important implications for understanding virion formation, virus interactions with host cells, and virus neutralization.