bluuebunny/crossref_metadata_2025 huggingface dataset:
The rat liver peroxisomal membrane forms a permeability barrier for cofactors but not for small metabolites in vitro
Authors: Antonenkov Vasily D. , Sormunen Raija T. , Hiltunen J. Kalervo
Published: October 2004

The functional role of the peroxisomal membrane as a permeability barrier to metabolites has been a matter of controversy for more than four decades. The initial conception, claiming free permeability of the membrane to small solute molecules, has recently been challenged by several observations suggesting that the peroxisomal membrane forms a closed compartment. We have characterized in vitro the permeability of rat liver peroxisomal membrane. Our results indicate that the membrane allows free access into peroxisomes for small hydrophilic molecules, such as substrates for peroxisomal enzymes (glycolate, urate), but not to more bulky cofactors (NAD/H, NADP/H, CoA). Although access for cofactors is not prevented completely by the membrane, the membrane barrier severely restricts their rate of entry into peroxisomes. The data lead to conclusion that, in vivo, peroxisomes may possess their own pool of cofactors, while they share a common pool of small metabolites with the cytoplasm. The results also indicate that molecular size plays an important role in in vivo distinction between cofactors and metabolic intermediates.
Application of Fluorescence Correlation Spectroscopy for Velocity Imaging in Microfluidic Devices
Authors: Kuricheti Kalyan K. , Buschmann Volker , Weston Kenneth D.
Published: October 2004

In this paper we present and demonstrate a technique for mapping fluid flow rates in microfluidic systems with sub-micrometer resolution using confocal microscopy in conjunction with fluorescence correlation spectroscopy (FCS). Flow velocities ranging from ∼50 μm/s to ∼ 10 cm/s can be recorded using fluorescent polymer nanospheres as fluid motion tracers. Velocity profiles and images of the flow in poly(dimethylsiloxane)–glass microchannels are presented and analyzed. Using the method, velocity images along the horizontal (top view) and vertical planes within a microdevice can be obtained. This is, to our knowledge, the first report of FCS for producing velocity maps. The high-resolution velocity maps can be used to characterize and optimize microdevice performance and to validate simulation efforts.
Prediction of Ethylene Content in Melt-State Random and Block Polypropylene by Near-Infrared Spectroscopy and Chemometrics: Comparison of a New Calibration Transfer Method with a Slope/Bias Correction Method
Authors: Watari Masahiro , Ozaki Yukihiro
Published: October 2004

This paper reports the prediction of the ethylene content (C2 content) in random polypropylene (RPP) and block polypropylene (BPP) in the melt state by near-infrared (NIR) spectroscopy and chemometrics. NIR spectra of RPP and BPP in the melt states were measured by a Fourier transform near-infrared (FT-NIR) on-line monitoring system. The NIR spectra of RPP and BPP were compared. Partial least-squares (PLS) regression calibration models predicting the ethylene (C2) content that were developed by using each RPP or BPP spectra set separately yielded good results (SECV (standard error of cross validation): RPP, 0.16%; BPP, 0.31%; correlation coefficient: RPP, 0.998; BPP, 0.996). We also built a common PLS calibration model by using both the RPP and the BPP spectra set. The results showed that the common calibration model has larger SECV values than the models based on the RPP or the BPP spectra sets individually and is not practical for the prediction of the C2 content. We further investigated whether a calibration model developed by using the BPP spectra set can predict the C2 contents in the RPP sample set. If this is possible, it can save a significant amount of work and cost. The results showed that the use of the BPP model for the RPP sample set is difficult, and vice versa, because there are some differences in the molar absorption coefficients between the RPP and BPP spectra. To solve this problem, a transfer method from one sample spectra (BPP) set to the other spectra (RPP) set was studied. A difference spectrum between an RPP spectrum and a BPP spectrum was used to transfer from the BPP calibration set to the RPP calibration set. The prediction result (SEP (standard error of prediction), 0.23%, correlation coefficient, 0.994) of RPP samples by the transferred calibration set and model showed that it is possible to transfer from the BPP calibration set to the RPP calibration set. We also studied the transfer from the RPP calibration set (the range of C2 content: 0–4.3%) to the BPP calibration set. The prediction result of C2 content (the range of C2 contents: 0–7.7%) in BPP by use of the calibration model based on the transferred BPP spectra from the RPP spectra showed that the transfer method is only effective for the interpolation of the C2 content range by the nonlinear change in the peak intensities with the C2 content.
Fourier Transform Infrared Imaging of Stereoregular Poly(Methyl Methacrylate) Dissolution
Authors: Bobiak J. P. , Koenig J. L.
Published: October 2004

Fourier transform infrared (FT-IR) imaging was used to study dissolution of stereoregular poly(methyl methacrylate) (PMMA) films in toluene, benzene, chloroform, acetic acid, and 2-ethoxyethanol. Images of the polymer–solvent interface showed that syndiotactic (sPMMA), atactic (aPMMA), and isotactic (iPMMA) samples dissolved in acetic acid and chloroform; only iPMMA dissolved in benzene, toluene, and 2-ethoxyethanol. Concentration profiles allowed quantitative comparison of each polymer–solvent system. In the cases of chloroform and acetic acid, the rate of polymer dissolution increased with increasing isotacticity. Dissolution of PMMA in chloroform was observed to coincide with development of strong polymer–solvent interactions. The results of systems containing toluene, benzene, and 2-ethoxyethanol reflect kinetic effects on dissolution, including solvent size and stiffness of the polymer backbone.
Three Novel Mutations in the Apolipoprotein E Gene in a Sample of Individuals with Type 2 Diabetes Mellitus
Authors: Stephens Jeffrey W , Sozen M Mert , Whittall Ros A , Caslake Muriel J , Bedford Dorothy , Acharya Jayshree , Hurel Steven J , Humphries Steve E
Published: October 2004

Background: Apolipoprotein E (apoE) is found in association with triglyceride-rich lipoproteins and is the ligand for the removal of these particles from the plasma. Genetic variations in exon 4 lead to three common gene variants: E2, E3, and E4.

Methods: We performed apoE genotyping in 765 individuals with type 2 diabetes.

Results: We identified three new variant heteroduplex patterns. Sequencing of these variants revealed three novel mutations that were related to biochemical and clinical characteristics. One mutation produced a frameshift at amino acid position 166, which predicted termination of protein synthesis. This individual had a heteroduplex pattern and sequence of E3E3, which was associated with a change in the plasma isoelectric focusing pattern and a 70% lower plasma concentration of apoE compared with healthy individuals. The other mutations were both single base changes. A CGC>CAC change at amino acid position 150 predicted a substitution of Arg>His. This individual had a heteroduplex pattern and sequence of E2E2, which was not associated with major changes in plasma lipids or apoE concentration. The third individual had a CGC>CCC base change at amino acid position 114, which predicted an Arg>Pro change. This person had a heteroduplex pattern and sequence of E3E3, higher plasma total cholesterol, and moderately decreased plasma apoE.

Conclusions: The frequency of new mutations in this sample (1 in 255) is higher than that of a healthy population (1 in 7900). Further screening for common apoE gene variants in individuals at risk for dyslipidemia may reveal abnormal heteroduplex patterns and uncover further mutations in this important lipid-regulating gene.
Fibroblast Growth Factor Receptor Signaling Promotes Radial Glial Identity and Interacts with Notch1 Signaling in Telencephalic Progenitors
Authors: Yoon Keejung , Nery Susana , Rutlin Michael L. , Radtke Freddy , Fishell Gord , Gaiano Nicholas
Published: October 2004

The Notch and fibroblast growth factor (FGF) pathways both regulate cell fate specification during mammalian neural development. We have shown previously that Notch1 activation in the murine forebrain promotes radial glial identity. This result, together with recent evidence that radial glia can be progenitors, suggested that Notch1 signaling might promote progenitor and radial glial character simultaneously. Consistent with this idea, we found that in addition to promoting radial glial characterin vivo, activated Notch1 (ActN1) increased the frequency of embryonic day 14.5 (E14.5) ganglionic eminence (GE) progenitors that grew into neurospheres in FGF2. Constitutive activation of C-promoter binding factor (CBF1), a Notch pathway effector, also increased neurosphere frequency in FGF2, suggesting that the effect of Notch1 on FGF responsiveness is mediated by CBF1.

The observation that ActN1 promoted FGF responsiveness in telencephalic progenitors prompted us to examine the effect of FGF pathway activationin vivo. We focused on FGFR2 because it is expressed in radial glia in the GEs where ActN1 increases FGF2 neurosphere frequency, but not in the septum where it does not. Like ActN1, activated FGFR2 (ActFGFR2) promoted radial glial characterin vivo. However, unlike ActN1, ActFGFR2 did not enhance neurosphere frequency at E14.5. Additional analysis demonstrated that, unexpectedly, neither ActFGFR2 nor ActFGFR1 could replace the need for ligand in promoting neurosphere proliferation. This study suggests that telencephalic progenitors with radial glial morphology are maintained by interactions between the Notch and FGF pathways, and that the mechanisms by which FGF signaling promotes radial glial characterin vivoand progenitor proliferationin vitrocan be uncoupled.
Brain Insulin Impairs Amyloid-β(1-40) Clearance from the Brain
Authors: Shiiki Takeshi , Ohtsuki Sumio , Kurihara Atsushi , Naganuma Hideo , Nishimura Kenji , Tachikawa Masanori , Hosoya Ken-ichi , Terasaki Tetsuya
Published: October 2004

Cerebral amyloid-β peptide (Aβ) clearance plays a key role in determining the brain level of Aβ; however, its mechanism remains unclear. In this study, we investigated cerebral Aβ clearance across the blood-brain barrier (BBB) by using the Brain Efflux Index method. [125I]Aβ(1-40) was eliminated from rat brain to circulating blood with a half-life of 48.8 min and a half-saturation concentration of 8.15 nm. The Aβ(1-40) elimination rate was reduced by 30.5% in 23-month-old rats compared with 7-week-old rats. The intact form of Aβ(1-40) was detected in plasma after intracerebral administration, indicating the occurrence of efflux transport of intact Aβ(1-40). The Aβ(1-40) elimination rate was significantly inhibited by coadministration of 100 μg/ml insulin and 1 mmthiorphan by 44.6 and 34.0%, respectively. The level of intact [125I]Aβ(1-40) in the brain was increased by coadministration of insulin. Among insulin-degrading enzyme inhibitors, bacitracin inhibited the elimination rate, whereasN-ethylmaleimide and metal chelators had no effect. Receptor-associated protein, fucoidan, 3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile, anti-IGF-I receptor antibody, andl-tyrosine did not affect the Aβ(1-40) elimination rate, suggesting that the relevant receptors or transporters are not likely to be involved in the clearance. In conclusion, the present study has demonstrated the involvement of a proteolytic degradation process and an insulin-sensitive process in cerebral Aβ(1-40) clearance in the rat.
Filamin A and FILIP (Filamin A-Interacting Protein) Regulate Cell Polarity and Motility in Neocortical Subventricular and Intermediate Zones during Radial Migration
Authors: Nagano Takashi , Morikubo Soichi , Sato Makoto
Published: October 2004

In the developing neocortex, most excitatory neurons are supplied and arranged through radial migration. Because neurons show global morphological changes and complicated behavior during that migration, precise regulation of cell shape and polarity is essential for proper migration and correct neocortical formation; however, how cell shape and polarity are regulated in migrating neuron remains elusive. We show here that Filamin A, a well known actin-binding protein, determines the shape of neocortical neurons during radial migrationin vivo. Dysfunction of Filamin A, caused by a mutant Filamin A expression, prevents cells from acquiring consistent polarity toward specific direction and decreases motility in the subventricular and intermediate zones. In contrast, Filamin A overexpression, achieved by a short interfering RNA for Filamin A-interacting protein that induces Filamin A degradation (FILIP), promotes the development and maintenance of a bipolar shape also in the subventricular and intermediate zones. These results suggest that the amount of Filamin A helps migrating neurons determine their mode of migration, multipolar or bipolar, before entering the cortical plate and that FILIP is responsible, at least in part, for Filamin A content. In addition, our results also give a possible clue to understanding the pathogenesis of human malformation periventricular heterotopia, which is caused by various “loss-of-function” mutations in thefilamin Agene.
Cell Type-Specific Synaptic Dynamics of Synchronized Bursting in the Juvenile CA3 Rat Hippocampus
Authors: Aradi Ildiko , Maccaferri Gianmaria
Published: October 2004

Spontaneous synchronous bursting of the CA3 hippocampusin vitrois a widely studied model of physiological and pathological network synchronization. The role of inhibitory conductances during network bursting is not understood in detail, despite the fact that several antiepileptic drugs target GABAAreceptors. Here, we show that the first manifestation of a burst event is a cell type-specific flurry of GABAAreceptor-mediated inhibitory input to pyramidal cells, but not to stratum oriens horizontal interneurons. Moreover, GABAAreceptor-mediated synaptic input is proportionally smaller in these interneurons compared with pyramidal cells. Computational models and dynamic-clamp studies using experimentally derived conductance waveforms indicate that both these factors modulate spike timing during synchronized activity. In particular, the different kinetics and the larger strength of GABAergic input to pyramidal cells defer action potential initiation and contribute to the observed delay of firing, so that the interneuronal activity leads the burst cycle. In contrast, excitatory inputs to both neuronal populations during a burst are kinetically similar, as required to maintain synchronicity. We also show that the natural pattern of activation of inhibitory and excitatory conductances during a synchronized burst cycle is different within the same neuronal population. In particular, GABAAreceptor-mediated currents activate earlier and outlast the excitatory components driving the bursts. Thus, cell type-specific balance and timing of GABAAreceptor-mediated input are critical to set the appropriate spike timing in pyramidal cells and interneurons and coordinate additional neurotransmitter release modulating burst strength and network frequency.
Human Immunodeficiency Virus Type 1 gp120 Induces Apoptosis in Human Primary Neurons through Redox-Regulated Activation of Neutral Sphingomyelinase
Authors: Jana Arundhati , Pahan Kalipada
Published: October 2004

Human immunodeficiency virus type 1 (HIV-1) infection is known to cause disorders of the CNS, including HIV-associated dementia (HAD). HIV-1 coat protein gp120 (glycoprotein 120) induces neuronal apoptosis and has been implicated in the pathogenesis of HAD. However, the mechanism by which gp120 causes neuronal apoptosis is poorly understood. The present study underlines the importance of gp120 in inducing the production of ceramide, an important inducer of apoptosis, in human primary neurons. gp120 induced the activation of sphingomyelinases (primarily the neutral one) and the production of ceramide in primary neurons. Antisense knockdown of neutral (NSMase) but not acidic (ASMase) sphingomyelinase markedly inhibited gp120-mediated apoptosis and cell death of primary neurons, suggesting that the activation of NSMase but not ASMase plays an important role in gp120-mediated neuronal apoptosis. Similarly, the HIV-1 regulatory protein Tat also induced neuronal cell death via NSMase. Furthermore, gp120-induced production of ceramide was redox sensitive, because reactive oxygen species were involved in the activation of NSMase but not ASMase. gp120 coupled CXCR4 (CXC chemokine receptor 4) to induce NADPH oxidase-mediated production of superoxide radicals in neurons, which was involved in the activation of NSMase but not ASMase. These studies suggest that gp120 may induce neuronal apoptosis in the CNS of HAD patients through the CXCR4-NADPH oxidase-superoxide-NSMase-ceramide pathway.
Extrasynaptic Localization of Inactivating Calcium-Activated Potassium Channels in Mouse Inner Hair Cells
Authors: Pyott Sonja J. , Glowatzki Elisabeth , Trimmer James S. , Aldrich Richard W.
Published: October 2004

Auditory hair cells from nonmammalian vertebrates are electrically tuned to specific sound frequencies primarily by the interactions of voltage-gated calcium channels and calcium-activated potassium (BK) channels colocalized at synaptic active zones. Mammalian inner hair cells are not electrically tuned and, yet, BK channels are also thought to reside at active zones. Using patch-clamp recordings and immunofluorescence, we characterized BK channel expression in mouse inner hair cells. Unexpectedly, these channels have inactivating currents and are clustered near the apex of the cell away from synaptic sites near the base. These results indicate a novel function of BK channels in mammalian inner hair cells and provide a framework for future research.
Heterosynaptic Long-Term Potentiation of Inhibitory Interneurons in the Lateral Amygdala
Authors: Bauer Elizabeth P. , LeDoux Joseph E.
Published: October 2004

Long-term potentiation (LTP) of synaptic transmission in the lateral amygdala (LA) is believed to underlie the formation and retention of fear memories. To explore the role of inhibitory transmission in amygdala plasticity, we recorded from LA inhibitory interneuronsin vitrobefore and after tetanization of the thalamo-LA pathway, one of the major inputs to LA involved in fear learning. Tetanization resulted in LTP of the EPSPs elicited in both the tetanized thalamic pathway and the untetanized cortical pathway to LA. This LTP was NMDA-dependent and associated with a decrease in paired-pulse facilitation in both pathways. In LA excitatory cells, LTP of interneurons resulted in an increase in the amplitude of GABAergic IPSPs in both input pathways. Finally, isolated GABAergic IPSPs between inhibitory and excitatory neurons could be potentiated as well. Plasticity of inhibitory transmission within the LA may therefore contribute significantly to LA-mediated functions, such as fear conditioning.
Calcium Release from Presynaptic Ryanodine-Sensitive Stores Is Required for Long-Term Depression at Hippocampal CA3-CA3 Pyramidal Neuron Synapses
Authors: Unni Vivek K. , Zakharenko Stanislav S. , Zablow Leonard , DeCostanzo Anthony J. , Siegelbaum Steven A.
Published: October 2004

Although Ca2+release from internal stores has been proposed to be important for the induction of long-term synaptic plasticity, the importance of Ca2+stores localized in presynaptic terminals remains unclear. Here, we have selectively applied pharmacological antagonists to either the presynaptic or postsynaptic cell in paired whole-cell recordings from hippocampal CA3 pyramidal neurons in slice culture. We demonstrate directly the necessary role of presynaptic, but not postsynaptic, ryanodine-sensitive Ca2+stores in the induction of NMDA receptor (NMDAR)-dependent long-term depression (LTD). Using two-photon laser scanning microscopy, we further find that release from the ryanodine-sensitive stores during prolonged synaptic stimulation generates a slowly rising Ca2+signal in the presynaptic terminal that is required for the induction of LTD. Moreover, this form of LTD has a significant presynaptic component of expression because it causes a marked decrease in the rate of release from CA3 neuron presynaptic terminals of FM 1-43, a fluorescent probe of synaptic vesicle cycling. Thus, Ca2+release from presynaptic ryanodine-sensitive stores is critical in the induction of a presynaptic component of NMDAR-dependent LTD.
Geometrische Normale zur Prüfung von Konturmessgeräten (Geometrical Standards for Testing of Contour Measuring Devices)
Authors: Neugebauer Michael , Jusko Otto , Wäldele Franz , Kedziora Heinz-Joachim , Meyer Maik , Volk Raimund
Published: October 2004

Es wird ein neu entwickeltes geometrisches Normal zur Prüfung von Konturmessgeräten im Tastschnittverfahren beschrieben. Dieses Arbeitsnormal vereint konvexe und konkave Geometrieelemente und verkörpert über 25 Radien, Winkel, laterale und vertikale Abstände. Die Kalibrierung erfolgt derzeit mit Koordinatenmessgeräten in der PTB. Die Kalibrierunsicherheiten betragen dabei 1 bis 1,5 μm für Abstände und Radien sowie 0,02° für Winkel. Für geplante Kalibrierungen mit Konturmessgeräten wurde zur Rückverfolgbarkeit der Messungen zusätzlich ein Transfernormal entwickelt.
Online-Analyse mit Lab-on-Chip-Systemen (Online Analysis with Lab-on-Chip Systems)
Authors: Herrmann Sigrun , Vonau Winfried
Published: October 2004

Es werden Ergebnisse der Entwicklung und Erprobung unterschiedlicher Lab-on-Chip-Systeme auf der Basis von elektrochemischen Sensoren vorgestellt. Das Design und der technologisch anspruchsvolle Herstellungsprozess von Sensorchips, aber auch Möglichkeiten der Verkapselung werden beschrieben. Die Entwicklung planarer Mikrosensorsysteme eröffnet beispielsweise die Möglichkeit zur Online-Analyse von Körperflüssigkeiten und zur Charakterisierung von Vitalitätskriterien. Anhand der ausgewählten Analysenbeispiele werden das breite Spektrum prinzipieller Einsatzmöglichkeiten für Lab-on-Chip-Systeme, gleichzeitig jedoch auch die Bedeutung der Materialauswahl für die Realisierung sensorspezifischer Anforderungen und applikationsbezogener Erfordernisse, wie z.B. Bio- oder Zellkompatibilität, deutlich.
Lrg1p Is a Rho1 GTPase-Activating Protein Required for Efficient Cell Fusion in Yeast
Authors: Fitch Pamela G , Gammie Alison E , Lee Debbie J , de Candal Valeria Brizzio , Rose Mark D
Published: October 2004

To identify additional cell fusion genes in Saccharomyces cerevisiae, we performed a high-copy suppressor screen of fus2Δ. Higher dosage of three genes, BEM1, LRG1, and FUS1, partially suppressed the fus2Δ cell fusion defect. BEM1 and FUS1 were high-copy suppressors of many cell-fusion-defective mutations, whereas LRG1 suppressed only fus2Δ and rvs161Δ. Lrg1p contains a Rho-GAP homologous region. Complete deletion of LRG1, as well as deletion of the Rho-GAP coding region, caused decreased rates of cell fusion and diploid formation comparable to that of fus2Δ. Furthermore, lrg1Δ caused a more severe mating defect in combination with other cell fusion mutations. Consistent with an involvement in cell fusion, Lrg1p localized to the tip of the mating projection. Lrg1p-GAP domain strongly and specifically stimulated the GTPase activity of Rho1p, a regulator of β(1-3)-glucan synthase in vitro. β(1-3)-glucan deposition was increased in lrg1Δ strains and mislocalized to the tip of the mating projection in fus2Δ strains. High-copy LRG1 suppressed the mislocalization of β(1-3) glucan in fus2Δ strains. We conclude that Lrg1p is a Rho1p-GAP involved in cell fusion and speculate that it acts to locally inhibit cell wall synthesis to aid in the close apposition of the plasma membranes of mating cells.
Meiotic Chromosome Synapsis in Yeast Can Occur Without Spo11-Induced DNA Double-Strand Breaks
Authors: Bhuiyan Hasanuzzaman , Schmekel Karin
Published: October 2004

Proper chromosome segregation and formation of viable gametes depend on synapsis and recombination between homologous chromosomes during meiosis. Previous reports have shown that the synaptic structures, the synaptonemal complexes (SCs), do not occur in yeast cells with the SPO11 gene removed. The Spo11 enzyme makes double-strand breaks (DSBs) in the DNA and thereby initiates recombination. The view has thus developed that synapsis in yeast strictly depends on the initiation of recombination. Synapsis in some other species (Drosophila melanogaster and Caenorhabditis elegans) is independent of recombination events, and SCs are found in spo11 mutants. This difference between species led us to reexamine spo11 deletion mutants of yeast. Using antibodies against Zip1, a SC component, we found that a small fraction (1%) of the spo11 null mutant cells can indeed form wild-type-like SCs. We further looked for synapsis in a spo11 mutant strain that accumulates pachytene cells (spo11Δ ndt80Δ), and found that the frequency of cells with apparently complete SC formation was 10%. Other phenotypic criteria, such as spore viability and homologous chromosome juxtaposition measured by FISH labeling of chromosomal markers, agree with several previous reports of the spo11 mutant. Our results demonstrate that although the Spo11-induced DSBs obviously promote synapsis in yeast, the presence of Spo11 is not an absolute requirement for synapsis.
Genetic Dissection of Nutritional Copper Signaling in Chlamydomonas Distinguishes Regulatory and Target Genes
Authors: Eriksson Mats , Moseley Jeffrey L , Tottey Stephen , del Campo Jose A , Quinn Jeanette , Kim Youngbae , Merchant Sabeeha
Published: October 2004

A genetic screen for Chlamydomonas reinhardtii mutants with copper-dependent growth or nonphotosynthetic phenotypes revealed three loci, COPPER RESPONSE REGULATOR 1 (CRR1), COPPER RESPONSE DEFECT 1 (CRD1), and COPPER RESPONSE DEFECT 2 (CRD2), distinguished as regulatory or target genes on the basis of phenotype. CRR1 was shown previously to be required for transcriptional activation of target genes like CYC6, CPX1, and CRD1, encoding, respectively, cytochrome c6 (which is a heme-containing substitute for copper-containing plastocyanin), coproporphyrinogen III oxidase, and Mg-protoporphyrin IX monomethylester cyclase. We show here that CRR1 is required also for normal accumulation of copper proteins like plastocyanin and ferroxidase in copper-replete medium and for apoplastocyanin degradation in copper-deficient medium, indicating that a single pathway controls nutritional copper homeostasis at multiple levels. CRR1 is linked to the SUPPRESSOR OF PCY1-AC208 13 (SOP13) locus, which corresponds to a gain-of-function mutation resulting in copper-independent expression of CYC6. CRR1 is required also for hypoxic growth, pointing to a physiologically meaningful regulatory connection between copper deficiency and hypoxia. The growth phenotype of crr1 strains results primarily from secondary iron deficiency owing to reduced ferroxidase abundance, suggesting a role for CRR1 in copper distribution to a multicopper ferroxidase involved in iron assimilation. Mutations at the CRD2 locus also result in copper-conditional iron deficiency, which is consistent with a function for CRD2 in a pathway for copper delivery to the ferroxidase. Taken together, the observations argue for a specialized copper-deficiency adaptation for iron uptake in Chlamydomonas.
Resistance to Volatile Anesthetics by Mutations Enhancing Excitatory Neurotransmitter Release in Caenorhabditis elegans
Authors: Hawasli Ammar H , Saifee Owais , Liu Christine , Nonet Michael L , Crowder C Michael
Published: October 2004

The molecular mechanisms whereby volatile general anesthetics (VAs) disrupt behavior remain undefined. In Caenorhabditis elegans mutations in the gene unc-64, which encodes the presynaptic protein syntaxin 1A, produce large allele-specific differences in VA sensitivity. UNC-64 syntaxin normally functions to mediate fusion of neurotransmitter vesicles with the presynaptic membrane. The precise role of syntaxin in the VA mechanism is as yet unclear, but a variety of results suggests that a protein interacting with syntaxin to regulate neurotransmitter release is essential for VA action in C. elegans. To identify additional proteins that function with syntaxin to control neurotransmitter release and VA action, we screened for suppressors of the phenotypes produced by unc-64 reduction of function. Loss-of-function mutations in slo-1, which encodes a Ca2+-activated K+ channel, and in unc-43, which encodes CaM-kinase II, and a gain-of-function mutation in egl-30, which encodes Gqα, were isolated as syntaxin suppressors. The slo-1 and egl-30 mutations conferred resistance to VAs, but unc-43 mutations did not. The effects of slo-1 and egl-30 on VA sensitivity can be explained by their actions upstream or parallel to syntaxin to increase the level of excitatory neurotransmitter release. These results strengthen the link between transmitter release and VA action.
Role of a Nonselective de Novo DNA Methyltransferase in Maternal Inheritance of Chloroplast Genes in the Green Alga, Chlamydomonas reinhardtii
Authors: Nishiyama Rie , Wada Yuko , Mibu Momoka , Yamaguchi Yube , Shimogawara Kosuke , Sano Hiroshi
Published: October 2004

In the green alga, Chlamydomonas, chloroplast DNA is maternally transmitted to the offspring. We previously hypothesized that the underlying molecular mechanism involves specific methylation of maternal gamete DNA before mating, protecting against degradation. To obtain direct evidence for this, we focused on a DNA methyltransferase, DMT1, which was previously shown to be localized in chloroplasts. The full-length DMT1 protein with a molecular mass of 150 kD was expressed in insect cells, and its catalytic activity was determined. In vitro assays using synthetic DNA indicated methylation of all cytosine residues, with no clear selectivity in terms of the neighboring nucleotides. Subsequently, transgenic paternal cells constitutively expressing DMT1 were constructed and direct methylation mapping assays of their DNA showed a clear nonselective methylation of chloroplast DNA. When transgenic paternal cells were crossed with wild-type maternal cells, the frequency of biparental and paternal offspring of chloroplasts increased up to 23% while between wild-type strains it was ∼3%. The results indicate that DMT1 is a novel type of DNA methyltransferase with a nonselective cytosine methylation activity, and that chloroplast DNA methylation by DMT1 is one of factors influencing maternal inheritance of chloroplast genes.
Accuracy and Power of Statistical Methods for Detecting Adaptive Evolution in Protein Coding Sequences and for Identifying Positively Selected Sites
Authors: Wong Wendy S W , Yang Ziheng , Goldman Nick , Nielsen Rasmus
Published: October 2004

The parsimony method of Suzuki and Gojobori (1999) and the maximum likelihood method developed from the work of Nielsen and Yang (1998) are two widely used methods for detecting positive selection in homologous protein coding sequences. Both methods consider an excess of nonsynonymous (replacement) substitutions as evidence for positive selection. Previously published simulation studies comparing the performance of the two methods show contradictory results. Here we conduct a more thorough simulation study to cover and extend the parameter space used in previous studies. We also reanalyzed an HLA data set that was previously proposed to cause problems when analyzed using the maximum likelihood method. Our new simulations and a reanalysis of the HLA data demonstrate that the maximum likelihood method has good power and accuracy in detecting positive selection over a wide range of parameter values. Previous studies reporting poor performance of the method appear to be due to numerical problems in the optimization algorithms and did not reflect the true performance of the method. The parsimony method has a very low rate of false positives but very little power for detecting positive selection or identifying positively selected sites.
Natural Genetic Variation Caused by Transposable Elements in Humans
Authors: Bennett E Andrew , Coleman Laura E , Tsui Circe , Pittard W Stephen , Devine Scott E
Published: October 2004

Transposons and transposon-like repetitive elements collectively occupy 44% of the human genome sequence. In an effort to measure the levels of genetic variation that are caused by human transposons, we have developed a new method to broadly detect transposon insertion polymorphisms of all kinds in humans. We began by identifying 606,093 insertion and deletion (indel) polymorphisms in the genomes of diverse humans. We then screened these polymorphisms to detect indels that were caused by de novo transposon insertions. Our method was highly efficient and led to the identification of 605 nonredundant transposon insertion polymorphisms in 36 diverse humans. We estimate that this represents 25–35% of ∼2075 common transposon polymorphisms in human populations. Because we identified all transposon insertion polymorphisms with a single method, we could evaluate the relative levels of variation that were caused by each transposon class. The average human in our study was estimated to harbor 1283 Alu insertion polymorphisms, 180 L1 polymorphisms, 56 SVA polymorphisms, and 17 polymorphisms related to other forms of mobilized DNA. Overall, our study provides significant steps toward (i) measuring the genetic variation that is caused by transposon insertions in humans and (ii) identifying the transposon copies that produce this variation.
A Workshop Report on Wheat Genome Sequencing
Authors: Gill Bikram S , Appels Rudi , Botha-Oberholster Anna-Maria , Buell C Robin , Bennetzen Jeffrey L , Chalhoub Boulos , Chumley Forrest , Dvořák Jan , Iwanaga Masaru , Keller Beat , Li Wanlong , McCombie W Richard , Ogihara Yasunari , Quetier Francis , Sasaki Takuji
Published: October 2004

Sponsored by the National Science Foundation and the U.S. Department of Agriculture, a wheat genome sequencing workshop was held November 10–11, 2003, in Washington, DC. It brought together 63 scientists of diverse research interests and institutions, including 45 from the United States and 18 from a dozen foreign countries (see list of participants at http://www.ksu.edu/igrow). The objectives of the workshop were to discuss the status of wheat genomics, obtain feedback from ongoing genome sequencing projects, and develop strategies for sequencing the wheat genome. The purpose of this report is to convey the information discussed at the workshop and provide the basis for an ongoing dialogue, bringing forth comments and suggestions from the genetics community.
Analysis of Expressed Sequence Tag Loci on Wheat Chromosome Group 4
Authors:   Miftahudin, Ross K , Ma X-F , Mahmoud A A , Layton J , Milla M A Rodriguez , Chikmawati T , Ramalingam J , Feril O , Pathan M S , Momirovic G Surlan , Kim S , Chema K , Fang P , Haule L , Struxness H , Birkes J , Yaghoubian C , Skinner R , McAllister J , Nguyen V , Qi L L , Echalier B , Gill B S , Linkiewicz A M , Dubcovsky J , Akhunov E D , Dvořák J , Dilbirligi M , Gill K S , Peng J H , Lapitan N L V , Bermudez-Kandianis C E , Sorrells M E , Hossain K G , Kalavacharla V , Kianian S F , Lazo G R , Chao S , Anderson O D , Gonzalez-Hernandez J , Conley E J , Anderson J A , Choi D-W , Fenton R D , Close T J , McGuire P E , Qualset C O , Nguyen H T , Gustafson J P
Published: October 2004

A total of 1918 loci, detected by the hybridization of 938 expressed sequence tag unigenes (ESTs) from 26 Triticeae cDNA libraries, were mapped to wheat (Triticum aestivum L.) homoeologous group 4 chromosomes using a set of deletion, ditelosomic, and nulli-tetrasomic lines. The 1918 EST loci were not distributed uniformly among the three group 4 chromosomes; 41, 28, and 31% mapped to chromosomes 4A, 4B, and 4D, respectively. This pattern is in contrast to the cumulative results of EST mapping in all homoeologous groups, as reported elsewhere, that found the highest proportion of loci mapped to the B genome. Sixty-five percent of these 1918 loci mapped to the long arms of homoeologous group 4 chromosomes, while 35% mapped to the short arms. The distal regions of chromosome arms showed higher numbers of loci than the proximal regions, with the exception of 4DL. This study confirmed the complex structure of chromosome 4A that contains two reciprocal translocations and two inversions, previously identified. An additional inversion in the centromeric region of 4A was revealed. A consensus map for homoeologous group 4 was developed from 119 ESTs unique to group 4. Forty-nine percent of these ESTs were found to be homoologous to sequences on rice chromosome 3, 12% had matches with sequences on other rice chromosomes, and 39% had no matches with rice sequences at all. Limited homology (only 26 of the 119 consensus ESTs) was found between wheat ESTs on homoeologous group 4 and the Arabidopsis genome. Forty-two percent of the homoeologous group 4 ESTs could be classified into functional categories on the basis of blastX searches against all protein databases.
A 2500-Locus Bin Map of Wheat Homoeologous Group 5 Provides Insights on Gene Distribution and Colinearity With Rice
Authors: Linkiewicz A M , Qi L L , Gill B S , Ratnasiri A , Echalier B , Chao S , Lazo G R , Hummel D D , Anderson O D , Akhunov E D , Dvořák J , Pathan M S , Nguyen H T , Peng J H , Lapitan N L V ,   Miftahudin, Gustafson J P , La Rota C M , Sorrells M E , Hossain K G , Kalavacharla V , Kianian S F , Sandhu D , Bondareva S N , Gill K S , Conley E J , Anderson J A , Fenton R D , Close T J , McGuire P E , Qualset C O , Dubcovsky J
Published: October 2004

We constructed high-density deletion bin maps of wheat chromosomes 5A, 5B, and 5D, including 2338 loci mapped with 1052 EST probes and 217 previously mapped loci (total 2555 loci). This information was combined to construct a consensus chromosome bin map of group 5 including 24 bins. A relatively higher number of loci were mapped on chromosome 5B (38%) compared to 5A (34%) and 5D (28%). Differences in the levels of polymorphism among the three chromosomes were partially responsible for these differences. A higher number of duplicated loci was found on chromosome 5B (42%). Three times more loci were mapped on the long arms than on the short arms, and a significantly higher number of probes, loci, and duplicated loci were mapped on the distal halves than on the proximal halves of the chromosome arms. Good overall colinearity was observed among the three homoeologous group 5 chromosomes, except for the previously known 5AL/4AL translocation and a putative small pericentric inversion in chromosome 5A. Statistically significant colinearity was observed between low-copy-number ESTs from wheat homoeologous group 5 and rice chromosomes 12 (88 ESTs), 9 (72 ESTs), and 3 (84 ESTs).
Deletion Mapping of Homoeologous Group 6-Specific Wheat Expressed Sequence Tags
Authors: Randhawa H S , Dilbirligi M , Sidhu D , Erayman M , Sandhu D , Bondareva S , Chao S , Lazo G R , Anderson O D ,   Miftahudin, Gustafson J P , Echalier B , Qi L L , Gill B S , Akhunov E D , Dvořák J , Linkiewicz A M , Ratnasiri A , Dubcovsky J , Bermudez-Kandianis C E , Greene R A , Sorrells M E , Conley E J , Anderson J A , Peng J H , Lapitan N L V , Hossain K G , Kalavacharla V , Kianian S F , Pathan M S , Nguyen H T , Endo T R , Close T J , McGuire P E , Qualset C O , Gill K S
Published: October 2004

To localize wheat (Triticum aestivum L.) ESTs on chromosomes, 882 homoeologous group 6-specific ESTs were identified by physically mapping 7965 singletons from 37 cDNA libraries on 146 chromosome, arm, and sub-arm aneuploid and deletion stocks. The 882 ESTs were physically mapped to 25 regions (bins) flanked by 23 deletion breakpoints. Of the 5154 restriction fragments detected by 882 ESTs, 2043 (loci) were localized to group 6 chromosomes and 806 were mapped on other chromosome groups. The number of loci mapped was greatest on chromosome 6B and least on 6D. The 264 ESTs that detected orthologous loci on all three homoeologs using one restriction enzyme were used to construct a consensus physical map. The physical distribution of ESTs was uneven on chromosomes with a tendency toward higher densities in the distal halves of chromosome arms. About 43% of the wheat group 6 ESTs identified rice homologs upon comparisons of genome sequences. Fifty-eight percent of these ESTs were present on rice chromosome 2 and the remaining were on other rice chromosomes. Even within the group 6 bins, rice chromosomal blocks identified by 1–6 wheat ESTs were homologous to up to 11 rice chromosomes. These rice-block contigs were used to resolve the order of wheat ESTs within each bin.
A Chromosome Bin Map of 2148 Expressed Sequence Tag Loci of Wheat Homoeologous Group 7
Authors: Hossain K G , Kalavacharla V , Lazo G R , Hegstad J , Wentz M J , Kianian P M A , Simons K , Gehlhar S , Rust J L , Syamala R R , Obeori K , Bhamidimarri S , Karunadharma P , Chao S , Anderson O D , Qi L L , Echalier B , Gill B S , Linkiewicz A M , Ratnasiri A , Dubcovsky J , Akhunov E D , Dvořák J ,   Miftahudin, Ross K , Gustafson J P , Radhawa H S , Dilbirligi M , Gill K S , Peng J H , Lapitan N L V , Greene R A , Bermudez-Kandianis C E , Sorrells M E , Feril O , Pathan M S , Nguyen H T , Gonzalez-Hernandez J L , Conley E J , Anderson J A , Choi D W , Fenton D , Close T J , McGuire P E , Qualset C O , Kianian S F
Published: October 2004

The objectives of this study were to develop a high-density chromosome bin map of homoeologous group 7 in hexaploid wheat (Triticum aestivum L.), to identify gene distribution in these chromosomes, and to perform comparative studies of wheat with rice and barley. We mapped 2148 loci from 919 EST clones onto group 7 chromosomes of wheat. In the majority of cases the numbers of loci were significantly lower in the centromeric regions and tended to increase in the distal regions. The level of duplicated loci in this group was 24% with most of these loci being localized toward the distal regions. One hundred nineteen EST probes that hybridized to three fragments and mapped to the three group 7 chromosomes were designated landmark probes and were used to construct a consensus homoeologous group 7 map. An additional 49 probes that mapped to 7AS, 7DS, and the ancestral translocated segment involving 7BS also were designated landmarks. Landmark probe orders and comparative maps of wheat, rice, and barley were produced on the basis of corresponding rice BAC/PAC and genetic markers that mapped on chromosomes 6 and 8 of rice. Identification of landmark ESTs and development of consensus maps may provide a framework of conserved coding regions predating the evolution of wheat genomes.
A Chromosome Bin Map of 16,000 Expressed Sequence Tag Loci and Distribution of Genes Among the Three Genomes of Polyploid Wheat
Authors: Qi L L , Echalier B , Chao S , Lazo G R , Butler G E , Anderson O D , Akhunov E D , Dvořák J , Linkiewicz A M , Ratnasiri A , Dubcovsky J , Bermudez-Kandianis C E , Greene R A , Kantety R , La Rota C M , Munkvold J D , Sorrells S F , Sorrells M E , Dilbirligi M , Sidhu D , Erayman M , Randhawa H S , Sandhu D , Bondareva S N , Gill K S , Mahmoud A A , Ma X-F ,   Miftahudin, Gustafson J P , Conley E J , Nduati V , Gonzalez-Hernandez J L , Anderson J A , Peng J H , Lapitan N L V , Hossain K G , Kalavacharla V , Kianian S F , Pathan M S , Zhang D S , Nguyen H T , Choi D-W , Fenton R D , Close T J , McGuire P E , Qualset C O , Gill B S
Published: October 2004

Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.
Chronology and Sequence of Eruption of the Permanent Teeth in Patients with Complete Unilateral Cleft Lip and Palate
Authors: Carrara Cleide Felíciode Carvalho , Lima JoséEduardode Oliveira , Carrara Carlos Eduardo , Vono Bernardo Gonzalez
Published: October 2004

Objective

To establish the chronology and sequence of eruption of the permanent teeth in subjects with complete unilateral cleft lip and palate.

Design

Cross-sectional. Data on children presenting complete cleft lip and palate were evaluated.

Setting

The study was carried out at the Hospital for Rehabilitation of Craniofacial Anomalies, Bauru, Sao Paulo, Brazil.

Sample

The sample comprised 477 patients with complete unilateral cleft lip and palate, aged 5 to 14 years. Of these patients, 166 were girls and 311 were boys.

Results

The girls presented, for all maxillary and mandibular teeth, a smaller mean age of eruption than the boys. The maxillary lateral incisor and cuspid adjacent to the cleft presented significantly higher mean ages of eruption than their homologous teeth on the noncleft side.
Cleft Lip and Palate in Jordan: Birth Prevalence Rate
Authors: Al Omari F. , Al-Omari I. K.
Published: October 2004

Objective and Design

A retrospective investigation was conducted to determine the prevalence rate of Jordanian children born with oral clefts from 1991 to 2001.

Setting

Hospital surgical records from two main sources were used to identify all children born with orofacial clefting. Information about date of birth, sex, cleft types, and associated major anomalies and syndromes were recorded.

Results

The overall prevalence rate for live births with cleft lip, cleft palate, or both was 1.39 per 1000 live births. Thirty percent of the clefts identified affected the lip, 22 percent affected the palate, and 48 percent involved the clefts of the lip and palate. In general, higher prevalence rates were found for boys than girls (55% boys versus 45% girls). There was a statistically significant sex difference between the cleft types. Clefts of the isolated palate, however, were more common in girls. Eighteen percent of patients with cleft were associated with major anomalies or syndromes.

Conclusions

The reported prevalence rate for orofacial clefts among Jordanians is similar to the previously reported prevalence rate in white Caucasians. Results and future recommendations are discussed.