bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrical characteristics of amorphous carbon nanotube and effects of contacts
Authors: Hu Y. F. , Liang X. L. , Chen Q. , Peng L.-M. , Hu Z. D.
Published: February 2006

Amorphous-carbon nanotube (a-CNT) based devices have been fabricated and transport measurements on these devices have been carried out. It was found that the Schottky barrier formed between the metal contact and a-CNT plays a significant role in the transport of a-CNT devices, and electrical characteristics of these devices may be engineered by varying the characteristics of the contact. In particular for asymmetrically contacted a-CNT devices it was demonstrated that the introduction of hydrogen may increase the current by more than 14 times at a positive bias of 3 V.
Shape and spatial correlation control of InAs-InAlAs-InP (001) nanostructure superlattices
Authors: Lei W. , Chen Y. H. , Jin P. , Ye X. L. , Wang Y. L. , Xu B. , Wang Z. G.
Published: February 2006

The control of shape and spatial correlation of InAs-InAlAs-InP(001) nanostructure superlattices has been realized by changing the As overpressure during the molecular-beam epitaxy (MBE) growth of InAs layers. InAs quantum wires (QWRs) are obtained under higher As overpressure (1×10−5Torr), while elongated InAs quantum dots (QDs) are formed under lower As overpressure (5×10−6 or 2.5×10−6Torr). Correspondingly, spatial correlation changes from vertical anti-correlation in QWR superlattices to vertical correlation in QD superlattices, which is well explained by the different alloy phase separation in InAlAs spacer layers triggered by the InAs nanostrcutures. It was observed that the alloy phase separation in QD superlattices could extend a long distance along the growth direction, indicating the vertical correlation of QD superlattices can be kept in a wide range of spacer layer thickness.
High-resolution spectral hole burning in InGaAs-GaAs quantum dots
Authors: Berry J. J. , Stevens Martin J. , Mirin R. P. , Silverman K. L.
Published: February 2006

We report the use of continuous wave spectral hole burning to perform high-resolution spectroscopy of the homogeneous linewidth of self-assembled InGaAs-GaAs quantum dots at low temperature. We use this technique to examine the power broadening behavior of the homogeneous InGaAs-GaAs quantum dot line. We find that at a temperature of 9.8 K and over the majority of the pump powers considered, the spectral hole signal is well fit by a single Lorentizian line shape. Analysis of the power broadening yields a full width at half maximum of 0.74μeV for the homogeneous linewidth and a corresponding coherence time T2 of 1.76 ns.
Nanogaps by direct lithography for high-resolution imaging and electronic characterization of nanostructures
Authors: Fischbein Michael D. , Drndić Marija
Published: February 2006

We report a method for fabricating nanogaps directly with electron beam lithography (EBL). The primary resolution-limit of EBL, electron back-scattering, is reduced dramatically by using a thin-film as a substrate. We show that this resolution enhancement allows one to fabricate metal electrodes with separation from arbitrarily large to under one nanometer. Furthermore, because these nanogaps are on a thin film, they can be imaged with high-resolution transmission electron microscopy (HRTEM). Using these nanogaps we measured the charge transport through several coupled PbSe nanocrystals and correlated the data with detailed structural information obtained by performing HRTEM on the same device.
Contrast in scanning probe microscopy images of ultrathin insulator films
Authors: Klust Andreas , Yu Qiuming , Olmstead Marjorie A. , Ohta Taisuke , Ohuchi Fumio S. , Bierkandt Markus , Deiter Carsten , Wollschläger Joachim
Published: February 2006

The contrast in scanning probe microscopy images of ultrathin CaF2 films epitaxially grown on Si(111) is studied using scanning tunneling microscopy (STM) and scanning force microscopy (SFM). Material contrast between CaF2 and the underlying Si–CaF interface layer can be achieved by multiple scan modes. STM images of CaF2∕Si(111) exhibit a distinct contrast depending on the bias voltage. Using SFM, CaF2 and CaF can be distinguished by the contact potential difference (CPD) determined by measuring the bias voltage dependence of the cantilever resonance frequency. The CPD also causes a material contrast in the dissipation signal.
Millimeter wave spectroscopy of rocks and fluids
Authors: Scales John A. , Batzle Michael
Published: February 2006

One region of the electromagnetic spectrum that is relatively unexploited for materials characterization is the millimeter wave band (frequencies roughly between 40 and 300GHz). Millimeter wave techniques involve free-space (noncontacting) measurements which have a length scale that makes them ideal for characterizing bulk properties of multicomponent composites where the scale of homogeneity is on the order of millimeters. Such composites include granular materials such as rocks, fluid mixtures, suspensions, and emulsions. Here we show measurements on partially saturated rocks and an oil/water mixture, demonstrating that millimeter wave spectroscopy is a sensitive yet rapid measure of changing composition.
Anisotropies of magnetic Compton profiles in Co∕Pd multilayer system
Authors: Sakurai H. , Ota M. , Itoh F. , Itou M. , Sakurai Y. , Koizumi A.
Published: February 2006

Anisotropies of spin-projected wave functions are measured on a Pd∕Co multilayer system by measuring magnetic Compton profiles. The anisotropies of the wave functions are decomposed into the contributions of Co 3d states and Pd 4d states using an atomic model by a Hartree-Fock calculation assuming uniaxial (cylindrical) symmetry. Perpendicular anisotropy in Pd∕Co multilayers is dominated by the anisotropy of Co 3d states; states with both magnetic quantum number ∣m∣=2 and ∣m∣=1 contribute.
Cyclin D1b variant influences prostate cancer growth through aberrant androgen receptor regulation
Authors: Burd Craig J. , Petre Christin E. , Morey Lisa M. , Wang Ying , Revelo Monica P. , Haiman Christopher A. , Lu Shan , Fenoglio-Preiser Cecilia M. , Li Jiwen , Knudsen Erik S. , Wong Jiemin , Knudsen Karen E.
Published: February 2006

Cyclin D1 is a multifaceted regulator of both transcription and cell-cycle progression that exists in two distinct isoforms, cyclin D1a and D1b. In the prostate, cyclin D1a acts through discrete mechanisms to negatively regulate androgen receptor (AR) activity and thus limit androgen-dependent proliferation. Accordingly, cyclin D1a is rarely overexpressed in prostatic adenocarcinoma and holds little prognostic value in this tumor type. However, a common polymorphism (A870) known to facilitate production of cyclin D1b is associated with increased prostate cancer risk. Here we show that cyclin D1b is expressed at high frequency in prostate cancer and is up-regulated in neoplastic disease. Furthermore, our data demonstrate that, although cyclin D1b retains AR association, it is selectively compromised for AR regulation. The altered ability of cyclin D1b to regulate the AR was observed by using both
in vitro
and
in vivo
assays and was associated with compromised regulation of AR-dependent proliferation. Consistent with previous reports, expression of cyclin D1a inhibited cell-cycle progression in AR-dependent prostate cancer cells. Strikingly, cyclin D1b significantly stimulated proliferation in this cell type. AR-negative prostate cancer cells were nonresponsive to cyclin D1 (a or b) expression, indicating that defects in AR corepressor function yield a growth advantage specifically in AR-dependent cells. In summary, these studies indicate that the altered AR regulatory capacity of cyclin D1b contributes to its association with increased prostate cancer risk and provide evidence of cyclin D1b-mediated transcriptional regulation.
S
-nitrosothiol depletion in amyotrophic lateral sclerosis
Authors: Schonhoff Christopher M. , Matsuoka Masaaki , Tummala Hemachand , Johnson Michael A. , Estevéz Alvaro G. , Wu Rui , Kamaid Andrés , Ricart Karina C. , Hashimoto Yuichi , Gaston Benjamin , Macdonald Timothy L. , Xu Zuoshang , Mannick Joan B.
Published: February 2006

Recent data suggest that either excessive or deficient levels of protein S-nitrosylation may contribute to disease. Disruption of
S
-nitrosothiol (SNO) homeostasis may result not only from altered nitric oxide (NO) synthase activity but also from alterations in the activity of denitrosylases that remove NO groups. A subset of patients with familial amyotrophic lateral sclerosis (ALS) have mutations in superoxide dismutase 1 (SOD1) that increase the denitrosylase activity of SOD1. Here, we show that the increased denitrosylase activity of SOD1 mutants leads to an aberrant decrease in intracellular protein and peptide S-nitrosylation in cell and animal models of ALS. Deficient S-nitrosylation is particularly prominent in the mitochondria of cells expressing SOD1 mutants. Our results suggest that SNO depletion disrupts the function and/or subcellular localization of proteins that are regulated by S-nitrosylation such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thereby contributes to ALS pathogenesis. Repletion of intracellular SNO levels with SNO donor compounds rescues cells from mutant SOD1-induced death. These results suggest that aberrant depletion of intracellular SNOs contributes to motor neuron death in ALS, and raises the possibility that deficient S-nitrosylation is a general mechanism of disease pathogenesis. SNO donor compounds may provide new therapeutic options for diseases such as ALS that are associated with deficient S-nitrosylation.
13
C isotopologue perturbation studies of
Listeria monocytogenes
carbon metabolism and its modulation by the virulence regulator PrfA
Authors: Eisenreich Wolfgang , Slaghuis Jörg , Laupitz Ralf , Bussemer Johanna , Stritzker Jochen , Schwarz Christine , Schwarz Roland , Dandekar Thomas , Goebel Werner , Bacher Adelbert
Published: February 2006

The carbon metabolism of
Listeria monocytogenes
(
Lm
) EGD and the two isogenic mutant strains
LmΔ
prfA and
Lm
Δ
prfA
pPRFA* (showing no or enhanced expression, respectively, of the virulence factor PrfA) was determined by
13
C isotopologue perturbation. After growth of the bacteria in a defined medium containing a mixture of [U-
13
C
6
]glucose and glucose with natural
13
C abundance (1:25, wt/wt), 14 amino acids were isolated and analyzed by NMR spectroscopy. Multiply
13
C-labeled isotopologues were determined quantitatively by signal deconvolution. The
13
C enrichments and isotopologue patterns allowed the reconstruction of most amino acid biosynthesis pathways and illustrated that overproduced PrfA may strongly influence the synthesis of some amino acids, notably that of the branched amino acids (Val, Ile, and Leu). Retrobiosynthetic analysis of the isotopologue compositions showed that degradation of glucose occurs to a large extent via the pentose phosphate pathway and that the citrate cycle is incomplete because of the absence of 2-oxoglutarate dehydrogenase activity. The reconstructed labeling pattern of oxaloacetate indicated its formation by carboxylation of pyruvate. This metabolic reaction seems to have a strong impact on the growth requirement in defined minimal medium. Bioinformatical steady-state network analyses and flux distribution predictions confirmed the experimental data and predicted metabolite fluxes through the enzymes of the pathways under study.
Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca
2+
and decreased cGMP-induced relaxation
Authors: Khromov A. S. , Wang H. , Choudhury N. , McDuffie M. , Herring B. P. , Nakamoto R. , Owens G. K. , Somlyo A. P. , Somlyo A. V.
Published: February 2006

Cyclic nucleotides can relax smooth muscle without a change in [Ca
2+
]
i
, a phenomenon termed Ca
2+
desensitization, contributing to vasodilation, gastrointestinal motility, and airway resistance. The physiological importance of telokin, a 17-kDa smooth muscle-specific protein and target for cyclic nucleotide-induced Ca
2+
desensitization, was determined in telokin null mice bred to a congenic background. Telokin null ileal smooth muscle homogenates compared to wild type exhibited an ≈30% decrease in myosin light-chain phosphatase (MLCP) activity, which was reflected in a significant leftward shift (up to 2-fold at pCa 6.3) of the Ca
2+
force relationship accompanied by an increase in myosin light-chain phosphorylation. No difference in the Ca
2+
force relationship occurred in telokin WT and knockout (KO) aortas, presumably reflecting the normally ≈5-fold lower telokin content in aorta vs. ileum smooth muscle. Ca
2+
desensitization of contractile force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal smooth muscle. The rate of force relaxation reflecting MLCP activity, in the presence of 50 μM 8-Br-cGMP, was also significantly slowed in telokin KO vs. WT ileum and was rescued by recombinant telokin. Normal thick filaments in telokin KO smooth muscles indicate that telokin is not required for filament formation or stability. Results indicate that a primary role of telokin is to modulate force through increasing MLCP activity and that this effect is further potentiated through phosphorylation by cGMP in telokin-rich smooth tissues.
Amplification of
MET
may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752
Authors: Smolen Gromoslaw A. , Sordella Raffaella , Muir Beth , Mohapatra Gayatry , Barmettler Anne , Archibald Heidi , Kim Woo J. , Okimoto Ross A. , Bell Daphne W. , Sgroi Dennis C. , Christensen James G. , Settleman Jeffrey , Haber Daniel A.
Published: February 2006

The success of molecular targeted therapy in cancer may depend on the selection of appropriate tumor types whose survival depends on the drug target, so-called “oncogene addiction.” Preclinical approaches to defining drug-responsive subsets are needed if initial clinical trials are to be directed at the most susceptible patient population. Here, we show that gastric cancer cells with high-level stable chromosomal amplification of the growth factor receptor MET are extraordinarily susceptible to the selective inhibitor PHA-665752. Although MET activation has primarily been linked with tumor cell migration and invasiveness, the amplified wild-type MET in these cells is constitutively activated, and its continued signaling is required for cell survival. Treatment with PHA-665752 triggers massive apoptosis in 5 of 5 gastric cancer cell lines with
MET
amplification but in 0 of 12 without increased gene copy numbers (
P
= 0.00016).
MET
amplification may thus identify a subset of epithelial cancers that are uniquely sensitive to disruption of this pathway and define a patient group that is appropriate for clinical trials of targeted therapy using MET inhibitors.
SAD-2 is required for meiotic silencing by unpaired DNA and perinuclear localization of SAD-1 RNA-directed RNA polymerase
Authors: Shiu Patrick K. T. , Zickler Denise , Raju Namboori B. , Ruprich-Robert Gwenael , Metzenberg Robert L.
Published: February 2006

A gene unpaired during the meiotic homolog pairing stage in
Neurospora
generates a sequence-specific signal that silences the expression of all copies of that gene. This process is called Meiotic Silencing by Unpaired DNA (MSUD). Previously, we have shown that SAD-1, an RNA-directed RNA polymerase (RdRP), is required for MSUD. We isolated a second gene involved in this process,
sad-2
. Mutated
Sad-2

RIP

alleles, like those of
Sad-1
, are dominant and suppress MSUD. Crosses homozygous for
Sad-2
are blocked at meiotic prophase. SAD-2 colocalizes with SAD-1 in the perinuclear region, where small interfering RNAs have been shown to reside in mammalian cells. A functional
sad-2
+
gene is necessary for SAD-1 localization, but the converse is not true. The data suggest that SAD-2 may function to recruit SAD-1 to the perinuclear region, and that the proper localization of SAD-1 is important for its activity.
Combinatorial
Gata2
and Sca1 expression defines hematopoietic stem cells in the bone marrow niche
Authors: Suzuki Norio , Ohneda Osamu , Minegishi Naoko , Nishikawa Mitsuo , Ohta Takayuki , Takahashi Satoru , Engel James Douglas , Yamamoto Masayuki
Published: February 2006

The interaction between stem cells and their supportive microenvironment is critical for their maintenance, function, and survival. Whereas hematopoietic stem cells (HSCs) are among the best characterized of tissue stem cells, their precise site of residence (referred to as the niche) in the adult bone marrow has not been precisely defined. In this study, we found that a
Gata2
promoter directs activity in all HSCs. We show that HSCs can be isolated efficiently from bone marrow cells by following
Gata2
-directed GFP fluorescence, and that they can also be monitored
in vivo
. Each individual GFP-positive cell lay in a G
0
/G
1
cell cycle state, in intimate contact with osteoblasts beside the endosteum, at the edge of the bone marrow. We conclude that the HSC niche is composed of solitary cells and that adult bone marrow HSC are not clustered.
The brain differentiates human and non-human grammars: Functional localization and structural connectivity
Authors: Friederici Angela D. , Bahlmann Jörg , Heim Stefan , Schubotz Ricarda I. , Anwander Alfred
Published: February 2006

The human language faculty has been claimed to be grounded in the ability to process hierarchically structured sequences. This human ability goes beyond the capacity to process sequences with simple transitional probabilities of adjacent elements observable in non-human primates. Here we show that the processing of these two sequence types is supported by different areas in the human brain. Processing of local transitions is subserved by the left frontal operculum, a region that is phylogenetically older than Broca’s area, which specifically holds responsible the computation of hierarchical dependencies. Tractography data revealing differential structural connectivity signatures for these two brain areas provide additional evidence for a segregation of two areas in the left inferior frontal cortex.
Role of positive selection in the retention of duplicate genes in mammalian genomes
Authors: Shiu Shin-Han , Byrnes Jake K. , Pan Runsun , Zhang Peng , Li Wen-Hsiung
Published: February 2006

The question of how duplicate genes are retained in a population remains controversial. The duplication-degeneration-complementation model, which involves no positive selection, stipulates a higher retention rate of duplicate genes in a small population than in a large one. This model has been accepted by many evolutionists. However, we found considerably more retentions and fewer losses of duplicate genes in the mouse genome than in the human genome, although the population size of rodents is in general larger than that of primates. Indeed, in nearly every interval of synonymous divergence between duplicate genes, the number of gene retentions in mouse is larger than that in human. Our findings suggest a more important role of positive selection in duplicate retention than duplication–degeneration–complementation. In addition, certain functional categories show a higher tendency of lineage-specific expansion than expected, suggesting lineage-specific selection or functional bias in retained duplicates.
Malaria risk on the Amazon frontier
Authors: de Castro Marcia Caldas , Monte-Mór Roberto L. , Sawyer Diana O. , Singer Burton H.
Published: February 2006

Frontier malaria is a biological, ecological, and sociodemographic phenomenon operating over time at three spatial scales (micro/individual, community, and state and national). We explicate these linkages by integrating data from remote sensing surveys, ground-level surveys and ethnographic appraisal, focusing on the Machadinho settlement project in Rondônia, Brazil. Spatially explicit analyses reveal that the early stages of frontier settlement are dominated by environmental risks, consequential to ecosystem transformations that promote larval habitats ofAnopheles darlingi. With the advance of forest clearance and the establishment of agriculture, ranching, and urban development, malaria transmission is substantially reduced, and risks of new infection are largely driven by human behavioral factors. Malaria mitigation strategies for frontier settlements require a combination of preventive and curative methods and close collaboration between the health and agricultural sectors. Of fundamental importance is matching the agricultural potential of specific plots to the economic and technical capacities of new migrants. Equally important is providing an effective agricultural extension service.
In vivo
repopulation ability of genetically corrected bone marrow cells from Fanconi anemia patients
Authors: Cohen-Haguenauer Odile , Péault Bruno , Bauche Cécile , Daniel Marie-Thérèse , Casal Ibrahim , Levy Vincent , Dausset Jean , Boiron Michel , Auclair Christian , Gluckman Eliane , Marty Michel
Published: February 2006

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction
ex vivo
, knockout animal models are not sufficient to guide preclinical steps, and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA, we could achieve long-term reconstitution of the stem cell compartment both
in vitro
and
in vivo
. Gene-corrected BM cultures grew for >120 days, and after cultured cell transplantation into NOD∕SCID mice, clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison, untransduced cells died in culture by 15 days. Of necessity for ethical reasons, experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements, a contingent of gene-corrected cells slowly emerges with an unmet potential for
in vivo
engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition, we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA.
The glycophorin C N-linked glycan is a critical component of the ligand for the
Plasmodium falciparum
erythrocyte receptor BAEBL
Authors: Mayer D. C. Ghislaine , Jiang Lubin , Achur Rajeshwara N. , Kakizaki Ikuko , Gowda D. Channe , Miller Louis H.
Published: February 2006

Plasmodium vivax
uses a single member of the Duffy binding-like (DBL) receptor family to invade erythrocytes and is not found in West Africa where its erythrocyte ligand, the Duffy blood group antigen, is missing. In contrast,
Plasmodium falciparum
expresses four members of the DBL family, and remarkably, single-point mutations of two of these receptors (BAEBL and JESEBL) bind to entirely different erythrocyte ligands, greatly expanding the range of erythrocytes that
P. falciparum
can invade. In this article, we describe the molecular basis of the binding specificity for one BAEBL variant (VSTK) that binds to glycophorin C. We demonstrate that soluble glycophorin C completely blocks the binding of BAEBL (VSTK) to human erythrocytes, requiring 0.7 μM for 50% inhibition, a concentration similar to that required by glycophorin A to block the binding of erythrocyte-binding antigen 175 to erythrocytes. BAEBL (VSTK) does not bind to Gerbich-negative erythrocytes that express a truncated form of glycophorin C because it lacks exon 3. The N-linked oligosaccharide of Gerbich-negative glycophorin C has a markedly different composition than the wild-type glycophorin C. Moreover, removal of the N-linked oligosaccharide from the wild-type glycophorin C eliminates its ability to inhibit binding of BAEBL (VSTK) to erythrocytes. These findings are consistent with the ligand for BAEBL (VSTK) being, in part, the N-linked oligosaccharide and suggest that single-point mutations in BAEBL allow
P. falciparum
to recognize oligosaccharides on different erythrocyte surface glycoproteins or glycolipids, greatly increasing its invasion range.
Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells
Authors: Yi MinKyung , Villanueva Rodrigo A. , Thomas David L. , Wakita Takaji , Lemon Stanley M.
Published: February 2006

Infections with hepatitis C virus (HCV) are marked by frequent viral persistence, chronic liver disease, and extraordinary viral genetic diversity. Although much has been learned about HCV since its discovery, progress has been slowed by a lack of permissive cell culture systems supporting its replication. Productive infections have been achieved recently with genotype 2a virus, but cirrhosis and liver cancer are typically associated with genotype 1 HCV, which is more prevalent and relatively resistant to IFN therapy. We describe production of infectious genotype 1a HCV in cells transfected with synthetic RNA derived from a prototype virus (H77-S). Viral proteins accumulated more slowly in H77-S transfected cells than in cells transfected with genotype 2a (JFH-1) RNA, but substantially more H77-S RNA was secreted into supernatant fluids. Most secreted RNA was noninfectious, banding in isopycnic gradients at a density of 1.04–1.07 gm∕cm
3
, but infectivity was associated with H77-S particles possessing a density of 1.13–1.14 gm∕cm
3
. The specific infectivity of H77-S particles (5.4 × 10
4
RNA copies per focus-forming unit) was significantly lower than JFH-1 virus (1.4 × 10
2
RNA copies per focus-forming unit). Infection with either virus was blocked by CD81 antibody. Sera from genotype 1a-infected individuals neutralized H77-S virus, but had little activity against genotype 2a virus, suggesting that these genotypes represent different serotypes. The ability of this genotype 1a virus to infect cultured cells will substantially benefit antiviral and vaccine discovery programs.
Evolution of enzymes in a series is driven by dissimilar functional demands
Authors: Salvador Armindo , Savageau Michael A.
Published: February 2006

That distinct enzyme activities in an unbranched metabolic pathway are evolutionarily tuned to a single functional requirement is a pervasive assumption. Here we test this assumption by examining the activities of two consecutively acting enzymes in human erythrocytes with an approach to quantitative evolutionary design that avoids the above-mentioned assumption. We previously found that avoidance of NADPH depletion during the pulses of oxidative load to which erythrocytes are normally exposed is the main functional requirement mediating selection for high glucose-6-phosphate dehydrogenase activity. In the present study, we find that, in contrast, the maintenance of oxidized glutathione at low concentrations is the main functional requirement mediating selection for high glutathione reductase activity. The results in this case show that, contrary to the assumption of a single functional requirement, natural selection for the normal activities of the distinct enzymes in the pathway is mediated by different requirements. On the other hand, the results agree with the more general principles that underlie our approach. Namely, that (i) the values of biochemical parameters evolve so as to fulfill the various performance requirements that are relevant to achieve high fitness, and (ii) these performance requirements can be inferred from quantitative systems theory considerations, informed by knowledge of specific aspects of the biochemistry, physiology, genetics, and ecology of the organism.
The unique cytoarchitecture of human pancreatic islets has implications for islet cell function
Authors: Cabrera Over , Berman Dora M. , Kenyon Norma S. , Ricordi Camillo , Berggren Per-Olof , Caicedo Alejandro
Published: February 2006

The cytoarchitecture of human islets has been examined, focusing on cellular associations that provide the anatomical framework for paracrine interactions. By using confocal microscopy and multiple immunofluorescence, we found that, contrary to descriptions of prototypical islets in textbooks and in the literature, human islets did not show anatomical subdivisions. Insulin-immunoreactive β cells, glucagon-immunoreactive α cells, and somatostatin-containing δ cells were found scattered throughout the human islet. Human β cells were not clustered, and most (71%) showed associations with other endocrine cells, suggesting unique paracrine interactions in human islets. Human islets contained proportionally fewer β cells and more α cells than did mouse islets. In human islets, most β, α, and δ cells were aligned along blood vessels with no particular order or arrangement, indicating that islet microcirculation likely does not determine the order of paracrine interactions. We further investigated whether the unique human islet cytoarchitecture had functional implications. Applying imaging of cytoplasmic free Ca
2+
concentration, [Ca
2+
]
i
, we found that β cell oscillatory activity was not coordinated throughout the human islet as it was in mouse islets. Furthermore, human islets responded with an increase in [Ca
2+
]
i
when lowering the glucose concentration to 1 mM, which can be attributed to the large contribution of α cells to the islet composition. We conclude that the unique cellular arrangement of human islets has functional implications for islet cell function.
Reprogramming of replicative senescence in hepatocellular carcinoma-derived cells
Authors: Ozturk Nuri , Erdal Esra , Mumcuoglu Mine , Akcali Kamil C. , Yalcin Ozden , Senturk Serif , Arslan-Ergul Ayca , Gur Bala , Yulug Isik , Cetin-Atalay Rengul , Yakicier Cengiz , Yagci Tamer , Tez Mesut , Ozturk Mehmet
Published: February 2006

Tumor cells have the capacity to proliferate indefinitely that is qualified as replicative immortality. This ability contrasts with the intrinsic control of the number of cell divisions in human somatic tissues by a mechanism called replicative senescence. Replicative immortality is acquired by inactivation of p53 and p16
INK4a
genes and reactivation of hTERT gene expression. It is unknown whether the cancer cell replicative immortality is reversible. Here, we show the spontaneous induction of replicative senescence in p53-and p16
INK4a
-deficient hepatocellular carcinoma cells. This phenomenon is characterized with hTERT repression, telomere shortening, senescence arrest, and tumor suppression. SIP1 gene (
ZFHX1B
) is partly responsible for replicative senescence, because short hairpin RNA-mediated SIP1 inactivation released hTERT repression and rescued clonal hepatocellular carcinoma cells from senescence arrest.
Cardiac-specific overexpression of sarcolipin in phospholamban null mice impairs myocyte function that is restored by phosphorylation
Authors: Gramolini Anthony O. , Trivieri Maria G. , Oudit Gavin Y. , Kislinger Thomas , Li Wenping , Patel Mikin M. , Emili Andrew , Kranias Evangelia G. , Backx Peter H. , MacLennan David H.
Published: February 2006

Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca
2+
ATPase (SERCA2a) by direct binding and is superinhibitory if it binds as a binary complex with phospholamban (PLN). To demonstrate whether overexpression of SLN in the heart might impair cardiac function directly, transgenic (TG) mice with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope) were generated on a phospholamban (PLN) null (PLN KO) background. In NF-SLN TG/PLN KO cardiac microsomes, the apparent affinity of SERCA2a for Ca
2+
was decreased compared with non-TG littermate PLN KO hearts. Analyses of isolated NF-SLN/PLN KO cardiomyocytes revealed impaired cardiac contractility, reduced calcium transient peak amplitude, and slower decay kinetics compared to PLN KO animals. In these cardiomyocytes, isoproterenol restored calcium dynamics to the levels seen in PLN KO. Invasive hemodynamic and echocardiographic analyses of NF-SLN/PLN KO mouse cardiac muscle
in vivo
showed no direct effects of NF-SLN overexpression when compared to PLN KO mice. A possible mechanism for the lack of effects in the whole heart may be a responsiveness to phosphorylation because we determined that NF-SLN can be phosphorylated in cardiomyocytes in response to isoproterenol, and we provide evidence that serine/threonine kinase 16 is a kinase that can phosphorylate NF-SLN. Site-directed mutagenesis showed that SLN Thr-5 is the target site for this kinase. These data show that overexpression of NF-SLN can inhibit SERCA2a in the absence of PLN and that the inhibition of SERCA2a is correlated with impairment of contractility and calcium cycling in cardiomyocytes.
Solution structure of p53 core domain: Structural basis for its instability
Authors: Cañadillas José Manuel Pérez , Tidow Henning , Freund Stefan M. V. , Rutherford Trevor J. , Ang Hwee Ching , Fersht Alan R.
Published: February 2006

The 25-kDa core domain of the tumor suppressor p53 is inherently unstable and melts at just above body temperature, which makes it susceptible to oncogenic mutations that inactivate it by lowering its stability. We determined its structure in solution using state-of-the-art isotopic labeling techniques and NMR spectroscopy to complement its crystal structure. The structure was very similar to that in the crystal but far more mobile than expected. Importantly, we were able to analyze by NMR the structural environment of several buried polar groups, which indicated structural reasons for the instability. NMR spectroscopy, with its ability to detect protons, located buried hydroxyl and sulfhydryl groups that form suboptimal hydrogen-bond networks. We mutated one such buried pair, Tyr-236 and Thr-253 to Phe-236 and Ile-253 (as found in the paralogs p63 and p73), and stabilized p53 by 1.6 kcal/mol. We also detected differences in the conformation of a mobile loop that might reflect the existence of physiologically relevant alternative conformations. The effects of temperature on the dynamics of aromatic residues indicated that the protein also experiences several dynamic processes that might be related to the presence of alternative hydrogen-bond patterns in the protein interior. p53 appears to have evolved to be dynamic and unstable.
Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses
Authors: Chi Hongbo , Barry Sean P. , Roth Rachel J. , Wu J. Julie , Jones Elizabeth A. , Bennett Anton M. , Flavell Richard A.
Published: February 2006

Engagement of Toll-like receptors (TLRs) on macrophages leads to activation of the mitogen-activated protein kinases (MAPKs), which contribute to innate immune responses. MAPK activity is regulated negatively by MAPK phosphatases (MKPs). MKP-1, the founding member of this family of dual-specificity phosphatases, has been implicated in regulating lipopolysaccharide (LPS) responses, but its role in TLR-mediated immune responses
in vivo
has not been defined. Here, we show that mice deficient in MKP-1 were highly susceptible to endotoxic shock
in vivo
, associated with enhanced production of proinflammatory cytokines TNF-α and IL-6 and an anti-inflammatory cytokine, IL-10. We further examined the regulation and function of MKP-1 in macrophages, a major cell type involved in endotoxic shock. MKP-1 was transiently induced by TLR stimulation through pathways mediated by both myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-β (TRIF). MKP-1 deficiency led to sustained activation of p38 MAPK and c-Jun N-terminal kinase (JNK) in LPS-treated macrophages. In response to TLR signals, MKP-1-deficient macrophages produced 5- to 10-fold higher IL-10, which could be blocked by a p38 MAPK inhibitor. Thus, p38 MAPK plays a critical role in mediating IL-10 synthesis in TLR signaling. TNF-α was found to be more abundant in MKP-1-deficient macrophages within 2 hours of TLR stimulation, but its production was rapidly down-regulated by IL-10. Our studies demonstrate that MKP-1 attenuates the activities of p38 MAPK and JNK to regulate both pro- and anti-inflammatory cytokines in TLR signaling. These results highlight the complex mechanisms by which the MAPKs regulate innate immunity.
Lateral mobility of proteins in liquid membranes revisited
Authors: Gambin Y. , Lopez-Esparza R. , Reffay M. , Sierecki E. , Gov N. S. , Genest M. , Hodges R. S. , Urbach W.
Published: February 2006

The biological function of transmembrane proteins is closely related to their insertion, which has most often been studied through their lateral mobility. For >30 years, it has been thought that hardly any information on the size of the diffusing object can be extracted from such experiments. Indeed, the hydrodynamic model developed by Saffman and Delbrück predicts a weak, logarithmic dependence of the diffusion coefficient
D
with the radius
R
of the protein. Despite widespread use, its validity has never been thoroughly investigated. To check this model, we measured the diffusion coefficients of various peptides and transmembrane proteins, incorporated into giant unilamellar vesicles of 1-stearoyl-2-oleoyl-
sn
-glycero-3-phosphocholine (SOPC) or in model bilayers of tunable thickness. We show in this work that, for several integral proteins spanning a large range of sizes, the diffusion coefficient is strongly linked to the protein dimensions. A heuristic model results in a Stokes-like expression for
D
, (
D
∝ 1/
R
), which fits literature data as well as ours. Diffusion measurement is then a fast and fruitful method; it allows determining the oligomerization degree of proteins or studying lipid–protein and protein–protein interactions within bilayers.
A transgenic approach for RNA interference-based genetic screening in mice
Authors: Peng Shaohua , York J. Philippe , Zhang Pumin
Published: February 2006

Genetic screening is the most powerful method through which to uncover gene function. It has been applied very successfully in lower organisms but seldom attempted in mammalian species because of their long generation time. In this study, we exploit RNA interference (RNAi) for its potential use in genetic screening in mice. We show that RNAi-induced gene knockdown can be generated through introducing small hairpin RNA-expressing constructs into the mouse as transgenes via conventional pronuclear injection. The knockdown effect can be transmitted for many generations in these transgenic animals. In a small-scale screening for developmental defects in the kidney, we uncovered a potential role of
Id4
in the formation of the renal medulla. Our results demonstrate the feasibility of using RNAi for genetic screening in mice.
Reciprocal actions of REST and a microRNA promote neuronal identity
Authors: Conaco Cecilia , Otto Stefanie , Han Jong-Jin , Mandel Gail
Published: February 2006

MicroRNAs (miRNAs) are implicated in both tissue differentiation and maintenance of tissue identity. In most cases, however, the mechanisms underlying their regulation are not known. One brain-specific miRNA, miR-124a, decreases the levels of hundreds of nonneuronal transcripts, such that its introduction into HeLa cells promotes a neuronal-like mRNA profile. The transcriptional repressor, RE1 silencing transcription factor (REST), has a reciprocal activity, inhibiting the expression of neuronal genes in nonneuronal cells. Here, we show that REST regulates the expression of a family of miRNAs, including brain-specific miR-124a. In nonneuronal cells and neural progenitors, REST inhibits miR-124a expression, allowing the persistence of nonneuronal transcripts. As progenitors differentiate into mature neurons, REST leaves miR-124a gene loci, and nonneuronal transcripts are degraded selectively. Thus, the combined transcriptional and posttranscriptional consequences of REST action maximize the contrast between neuronal and nonneuronal cell phenotypes.
Fast complementation of split fluorescent protein triggered by DNA hybridization
Authors: Demidov Vadim V. , Dokholyan Nikolay V. , Witte-Hoffmann Carlos , Chalasani Poornima , Yiu Hung-Wei , Ding Feng , Yu Yong , Cantor Charles R. , Broude Natalia E.
Published: February 2006

Fluorescent proteins have proven to be excellent reporters and biochemical sensors with a wide range of applications. In a split form, they are not fluorescent, but their fluorescence can be restored by supplementary protein–protein or protein–nucleic acid interactions that reassemble the split polypeptides. However, in prior studies, it took hours to restore the fluorescence of a split fluorescent protein because the formation of the protein chromophore slowly occurred
de novo
concurrently with reassembly. Here we provide evidence that a fluorogenic chromophore can self-catalytically form within an isolated N-terminal fragment of the enhanced green fluorescent protein (EGFP). We show that restoration of the split protein fluorescence can be driven by nucleic acid complementary interactions. In our assay, fluorescence development is fast (within a few minutes) when complementary oligonucleotide-linked fragments of the split EGFP are combined. The ability of our EGFP system to respond quickly to DNA hybridization should be useful for detecting the kinetics of many other types of pairwise interactions both
in vitro
and in living cells.
Nanomechanics of the subtectorial space caused by electromechanics of cochlear outer hair cells
Authors: Nowotny Manuela , Gummer Anthony W.
Published: February 2006

The stereocilia of the cochlear inner hair cells (IHCs) transduce vibrations into the sensory receptor current. Until now, mechanisms for deflecting these stereocilia have not been identified experimentally. Here, we identify a mechanism by using the electromechanical properties of the soma of the outer hair cell to produce an intracochlear, mechanical force stimulus. It is known that the soma of this cell generates mechanical force in response to a change of its transmembrane potential. In the present experiments, the force was induced by intracochlear electrical stimulation at frequencies that covered the entire functionally relevant range of 50 kHz. Vibration responses were measured in the transverse direction with a laser Doppler vibrometer. For frequencies up to ≈3 kHz in the first three turns of the guinea-pig cochlea, the apical surface of the IHC and the opposing surface of the tectorial membrane were found to vibrate with similar amplitudes but opposite phases. At high frequencies, there was little relative motion between these surfaces in the transverse direction. The counterphasic motion up to ≈3 kHz results in a pulsatile motion of the fluid surrounding the stereocilia of the IHCs. Based on physical principles of fluid flow between narrowly spaced elastic plates, we show that radial fluid motion is amplified relative to transverse membrane motion and that the radial motion is capable of bending the stereocilia. In conclusion, for frequencies up to at least 3 kHz, there appears to be direct fluid coupling between outer hair cells and IHCs.
Core domain interactions in full-length p53 in solution
Authors: Veprintsev Dmitry B. , Freund Stefan M. V. , Andreeva Antonina , Rutledge Stacey E. , Tidow Henning , Cañadillas José Manuel Pérez , Blair Caroline M. , Fersht Alan R.
Published: February 2006

The tumor suppressor p53 consists of four 393-residue chains, each of which has two natively unfolded (N- and C-terminal) and two folded (core and tetramerization) domains. Their structural organization is poorly characterized as the protein tends to aggregate, has defied crystallization, and is at the limits of NMR studies. We first stabilized the protein by mutation to make it more suitable for extended study and then acquired NMR spectra on full-length protein and various combinations of shorter domain constructs. The NMR spectrum (
15
N,
1
H transverse relaxation optimized spectroscopy) of full-length p53 was close to that expected from the sum of the spectra of isolated individual domains. However, patterns of changes in chemical shifts revealed unexpected interactions between the core domains. We used the NMR data as constraints in docking algorithms and found a previously uncharacterized self-complementary surface for the association of core domains into dimers within the tetrameric complex. Binding to DNA requires about a 70° rotation to break those subunit interactions and form the known protein:protein interface in the p53–DNA complex. We verified the interactions by the effects of mutation on DNA binding. Spectroscopic, biophysical, and mutational data conspired to give a picture of the p53 tetramer as a dimer of loosely tethered core dimers of appropriate symmetry to be poised to bind target DNA.
Recommendations for the Interpretation of Renal Tubule Proliferative Lesions Occurring in Rat Kidneys with Advanced Chronic Progressive Nephropathy (CPN)
Authors: Hard Gordon C. , Seely John Curtis
Published: February 2006

There is little guidance in the literature on the spectrum of proliferative tubule lesions in the kidneys of aging rats affected with spontaneously occurring, chronic progressive nephropathy (CPN), or their interpretation. Through accessing 2-year carcinogenicity studies in male F344 rats held in the Archives of the National Toxicology Program, NIEHS, a large number of cases of advanced CPN have been surveyed histopathologically for proliferative tubule lesions, and an attempt made to provide guidelines for discrimination of lesions common to the CPN process, from those representing precursors of neoplasia. Several proliferative lesions were identified as common in advanced CPN with no apparent evidence supporting a role in renal tubule carcinogenesis. It is recommended that these lesions be viewed generically as CPN tubule profiles, and not recorded separately from the diagnosis of CPN. Criteria were developed to distinguish these CPN-associated lesions from atypical tubule hyperplasia, a precursor of adenoma, both of which were also represented in this survey of advanced CPN.
Oncogenic Signaling Pathways Activated in DMBA-Induced Mouse Mammary Tumors
Authors: Currier Nicolas , Solomon Sandra E. , Demicco Elizabeth G. , Chang Donny L. F. , Farago Marganit , Ying Haoqiang , Dominguez Isabel , Sonenshein Gail E. , Cardiff Robert D. , Xiao Zhi-Xiong Jim , Sherr David H. , Seldin David C.
Published: February 2006

Only about 5% of human breast cancers can be attributed to inheritance of breast cancer susceptibility genes, while the balance are considered to be sporadic in origin. Breast cancer incidence varies with diet and other environmental influences, including carcinogen exposure. However, the effects of environmental carcinogens on cell growth control pathways are poorly understood. Here we have examined oncogenic signaling pathways that are activated in mammary tumors in mice treated with the prototypical polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[ a]anthracene (DMBA). In female FVB mice given 6 doses of 1 mg of DMBA by weekly gavage beginning at 5 weeks of age, all of the mice developed tumors by 34 weeks of age (median 20 weeks after beginning DMBA); 75% of the mice had mammary tumors. DMBA-induced mammary tumors exhibited elevated expression of the aryl hydrocarbon receptor (AhR), c- myc, cyclin D1, and hyperphosphorylated retinoblastoma (Rb) protein. Because of this, the activation of upstream regulatory pathways was assessed, and elements of the Wnt signaling pathway, the NF-κB pathway, and the prolyl isomerase Pin-1 were found to be frequently up-regulated in the tumors when compared to normal mammary gland controls. These data suggest that environmental carcinogens can produce long-lasting alterations in growth and anti-apoptotic pathways, leading to mammary tumorigenesis.