bluuebunny/crossref_metadata_2025 huggingface dataset:
Vital pulp therapy for the mature tooth – can it work?
Authors: SWIFT EDWARD J. , TROPE MARTIN , RITTER ANDRÉ V.
Published: February 2004

The dental pulp can be exposed by accidental trauma to a tooth, or by the dentist preparing a tooth for a restoration. When the exposure occurs during cavity preparation, it can be diagnosed as mechanical or carious. The pulp typically is inflamed in either instance, because a mechanical exposure rarely occurs except during removal of a deep restoration or through overzealous excavation of deep caries. Pulpectomy and root canal therapy represent the optimal treatment for carious or mechanically exposed pulps in mature teeth. However, financial considerations or low dental IQ result in some patients refusing the optimal treatment. In our opinion, extraction is not the correct alternative treatment plan for these patients. This paper describes the indications, clinical techniques, and prognosis for three types of vital pulp therapy in the mature tooth – direct pulp capping, partial pulpotomy, and full pulpotomy.
The role of carbon monoxide in the gastrointestinal tract
Authors: Gibbons Simon J. , Farrugia Gianrico
Published: February 2004

Carbon monoxide (CO) is a biologically active product of haem metabolism that contributes to the normal physiology of the gastrointestinal tract. In this article, we review recent data showing that CO is an integral regulator of gastrointestinal motility and an important factor in the response to gastrointestinal injury. CO is generated by haem oxygenase‐2 (HO‐2), which is constitutively expressed in many inhibitory neurones of the vertebrate enteric nervous system. The membrane potential gradients along and across the muscle layers of the gastrointestinal tract require the generation of CO by haem oxygenase‐2. The presence of CO is also necessary for normal inhibitory neurotransmission in circular smooth muscle and appears to permit nitric oxide‐mediated inhibitory neurotransmission. Genetic deletion of the haem oxygenase‐2 gene in mice slows gut transit. The other major CO synthetic enzyme, haem oxygenase‐1 (HO‐1) is induced under conditions of stress or injury. Recent studies have demonstrated that up‐regulation of haem oxygenase‐1 protects the gut from several types of gastrointestinal injury, suggesting that CO or induction of HO‐1 may find therapeutic use in gastrointestinal diseases and injuries. Furthermore, it is anticipated that the understanding of CO‐mediated signalling in the gastrointestinal tract will inform studies in other tissues that express haem oxygenases.
Regulation of rat mesencephalic GABAergic neurones through muscarinic receptors
Authors: Michel François J. , Robillard Julie M. , Trudeau Louis‐Éric
Published: February 2004

Central dopamine neurones are involved in regulating cognitive and motor processes. Most of these neurones are located in the ventral mesencephalon where they receive abundant intrinsic and extrinsic GABAergic input. Cholinergic neurones, originating from mesopontine nuclei, project profusely in the mesencephalon where they preferentially synapse onto local GABAergic neurones. The physiological role of this cholinergic innervation of GABAergic neurones remains to be determined, but these observations raise the hypothesis that ACh may regulate dopamine neurones indirectly through GABAergic interneurones. Using a mesencephalic primary culture model, we studied the impact of cholinergic agonists on mesencephalic GABAergic neurones. ACh increased the frequency of spontaneous IPSCs (151 ± 49%). Selective activation of muscarinic receptors increased the firing rate of isolated GABAergic neurones by 67 ± 13%. The enhancement in firing rate was Ca2+ dependent since inclusion of BAPTA in the pipette blocked it, actually revealing a decrease in firing rate accompanied by membrane hyperpolarization. This inhibitory action was prevented by tertiapin, a blocker of GIRK‐type K+ channels. In addition to its excitatory somatodendritic effect, activation of muscarinic receptors also acted presynaptically, inhibiting the amplitude of unitary GABAergic synaptic currents. Both the enhancement in spontaneous IPSC frequency and presynaptic inhibition were abolished by 4‐DAMP (100 nm), a preferential M3 muscarinic receptor antagonist. The presence of M3‐like receptors on mesencephalic GABAergic neurones was confirmed by immunocytochemistry. Taken together, these results demonstrate that mesencephalic GABAergic neurones can be regulated directly through muscarinic receptors. Our findings provide new data that should be helpful in better understanding the influence of local GABAergic neurones during cholinergic activation of mesencephalic circuits.
Charged residue changes in the carboxy‐terminus of α‐tropomyosin alter mouse cardiac muscle contractility
Authors: Gaffin Robert D. , Gokulan Kuppan , Sacchettini James C. , Hewett Timothy , Klevitsky Raisa , Robbins Jeffrey , Muthuchamy Mariappan
Published: February 2004

Striated muscle tropomyosin (TM) is an essential thin filament protein that is sterically and allosterically involved in calcium‐mediated cardiac contraction. We have previously shown that overexpressing the β‐TM isoform in mouse hearts leads to physiological changes in myocardial relaxation and Ca2+ handling of myofilaments. Two important charge differences in β‐TM compared to α‐TM are the exchange of serine and histidine at positions 229 and 276 with glutamic acid and asparagine, respectively, imparting a more negative charge to β‐TM relative to α‐TM. Our hypothesis is that the net charge at specific sites on TM might be a major determinant of its role in modulating cardiac muscle performance and in regulating Ca2+ sensitivity of the myofilaments. To address this, we generated transgenic (TG) double mutation mouse lines (α‐TM DM) expressing mutated α‐TM at the two residues that differ between α‐ and β‐TM (Ser229Glu + His276Asn). Molecular analyses show 60–88% of the native TM is replaced with α‐TM DM in the different TG lines. Work‐performing heart analyses show that α‐TM DM mouse hearts exhibit decreased rates of pressure development and relaxation (+dP/dt and –dP/dt). Skinned myofibre preparations from the TG hearts indicate a decrease in calcium sensitivity of steady state force. Protein modelling studies show that these two charge alterations in α‐TM cause a change in the surface charges of the molecule. Our results provide the first evidence that charge changes at the carboxy‐terminal of α‐TM alter the functional characteristics of the heart at both the whole organ and myofilament levels.
Modulation of spontaneous Ca2+‐activated Cl− currents in the rabbit corpus cavernosum by the nitric oxide–cGMP pathway
Authors: Craven M. , Sergeant G. P. , Hollywood M. A. , McHale N. G. , Thornbury K. D.
Published: February 2004

The whole‐cell perforated patch clamp technique was used to study membrane currents in isolated rabbit corpus cavernosum smooth muscle cells. Depolarization from –80 mV to the range –40 to –10 mV evoked a nifedipine‐sensitive Ca2+ current that was followed by a slower inward current that activated over several hundred milliseconds. The slow current reversed near the Cl− equilibrium potential (ECl) and was reduced by anthracene‐9‐carboxylic acid (A9C; 1 mm) and niflumic acid (100 μm), suggesting that it was a Ca2+‐activated Cl− current. When held constantly at –60 mV, over 70% of cells fired spontaneous transient inward currents (STICs), the amplitudes of which were reduced by A9C and niflumic acid. STICs reversed near ECl in a symmetrical Cl− gradient and when [Cl−]o was substituted with glutamate or I−, the reversal potential shifted to more positive or more negative values, respectively, confirming that STICs were mediated by Cl− channels. STICS were also blocked by cyclopiazonic acid, 2‐aminoethoxydiphenyl borate (2‐APB) and 2‐nitro‐4‐carboxyl‐N,N‐diphenylcarbamate (NCDC), suggesting that they depended on IP3‐mediated Ca2+‐release from the sarcoplasmic reticulum. Modulation by the NO–cGMP pathway was investigated by applying nitrosocysteine, 3‐(5‐hydroxymethyl‐2‐furyl)‐1‐benzyl indazole (YC‐1), and 8‐bromo cGMP, all three of which abolished STIC activity. YC‐1 also reduced noradrenaline‐evoked inward currents, but had no effect on similar currents evoked by caffeine, suggesting that cGMP selectively inhibited IP3‐mediated Ca2+ release. We propose that Ca2+‐activated Cl− currents underlie detumescent tone in the corpus cavernosum, and that modulation of this mechanism by the NO–cGMP pathway is important during penile erection.
Human mesenchymal stem cells make cardiac connexins and form functional gap junctions
Authors: Valiunas Virginijus , Doronin Sergey , Valiuniene Laima , Potapova Irina , Zuckerman Joan , Walcott Benjamin , Robinson Richard B. , Rosen Michael R. , Brink Peter R. , Cohen Ira S.
Published: February 2004

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell‐to‐cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell‐to‐cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents (Ij) recorded between hMSC pairs exhibited quasi‐symmetrical and asymmetrical voltage (Vj) dependence. Ij records from hMSC–HeLaCx43 and hMSC–HeLaCx40 cell pairs also showed symmetrical and asymmetrical Vj dependence, while hMSC–HeLaCx45 pairs always produced asymmetrical Ij with pronounced Vj gating when the Cx45 side was negative. Symmetrical Ij suggests that the dominant functional channel is homotypic, while the asymmetrical Ij suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (γj) of 30–80pS. The macroscopic Ij obtained from hMSC–cardiac myocyte cell pairs exhibited asymmetrical Vj dependence, while single channel events revealed γj of the size range 40–100pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.
Mitochondrial modulation of Ca2+ sparks and transient KCa currents in smooth muscle cells of rat cerebral arteries
Authors: Cheranov Serguei Y. , Jaggar Jonathan H.
Published: February 2004

Mitochondria sequester and release calcium (Ca2+) and regulate intracellular Ca2+ concentration ([Ca2+]i) in eukaryotic cells. However, the regulation of different Ca2+ signalling modalities by mitochondria in smooth muscle cells is poorly understood. Here, we investigated the regulation of Ca2+ sparks, Ca2+ waves and global [Ca2+]i by mitochondria in cerebral artery smooth muscle cells. CCCP (a protonophore; 1 μm) and rotenone (an electron transport chain complex I inhibitor; 10 μm) depolarized mitochondria, reduced Ca2+ spark and wave frequency, and elevated global [Ca2+]i in smooth muscle cells of intact arteries. In voltage‐clamped (−40 mV) cells, mitochondrial depolarization elevated global [Ca2+]i, reduced Ca2+ spark amplitude, spatial spread and the effective coupling of sparks to large‐conductance Ca2+‐activated potassium (KCa) channels, and decreased transient KCa current frequency and amplitude. Inhibition of Ca2+ sparks and transient KCa currents by mitochondrial depolarization could not be explained by a decrease in intracellular ATP or a reduction in sarcoplasmic reticulum Ca2+ load, and occurred in the presence of diltiazem, a voltage‐dependent Ca2+ channel blocker. Ru360 (10 μm), a mitochondrial Ca2+ uptake blocker, and lonidamine (100 μm), a permeability transition pore (PTP) opener, inhibited transient KCa currents similarly to mitochondrial depolarization. In contrast, CGP37157 (10 μm), a mitochondrial Na+–Ca2+ exchange blocker, activated these events. The PTP blockers bongkrekic acid and cyclosporin A both reduced inhibition of transient KCa currents by mitochondrial depolarization. These results indicate that mitochondrial depolarization leads to a voltage‐independent elevation in global [Ca2+]i and Ca2+ spark and transient KCa current inhibition. Data also suggest that mitochondrial depolarization inhibits Ca2+ sparks and transient KCa currents via PTP opening and a decrease in intramitochondrial [Ca2+].
Aspartate transporter expression and activity in hypertrophic rat heart and ischaemia–reperfusion injury
Authors: King Nicola , Lin Hua , McGivan John D. , Suleiman M.‐Saadeh
Published: February 2004

This study's rationale was that the expression and activity of aspartate transporters in hypertrophied hearts might be different from normal hearts, which could affect the use of aspartate in myocardial protection of hypertrophied hearts. mRNA expression of system Xag− transporters in hearts from normal (Wistar Kyoto) and hypertrophied (spontaneously hypertensive rat) rats was investigated by RT‐PCR. EAAT3 protein expression in isolated cells and vesicles from normal and hypertrophied hearts was investigated by Western blotting. The same vesicles were also used to measure aspartate uptake. The effects of 0.5 mmol l−1 aspartate supplementation on cardiac performance during ischaemia–reperfusion were investigated in isolated and perfused hearts. Both normal and hypertrophied hearts expressed EAAT1 and EAAT3 mRNA. EAAT3 protein expression was significantly greater in cells and vesicles from hypertrophied hearts compared to normal hearts. The velocity (Vmax) of aspartate uptake was faster at 24.4 ± 2.2 pmol mg−1 s−1 in vesicles from hypertrophied hearts compared to 8.2 ± 0.8 pmol mg−1 s−1 (P < 0.001, t test, n= 6, means ±s.e.m.) in normal heart vesicles. The affinity (Km) was similar for both preparations. When recoveries were matched, 0.5 mmol l−1 aspartate addition reduced reperfusion injury and increased functional recovery of hypertrophied hearts but not normal hearts. This was associated with a greater preservation of ATP, glutamate and glutamine and less lactate production during ischaemia in aspartate‐treated hypertrophied hearts compared to all other experimental groups. These results suggest that increased aspartate transporter expression and activity in hypertrophy helps facilitate aspartate entry into hypertrophied cardiomyocytes, which in turn leads to improved myocardial protection.
Afferent modulation of neonatal rat respiratory rhythm in vitro: cellular and synaptic mechanisms
Authors: Mellen Nicholas M. , Roham Maryam , Feldman Jack L.
Published: February 2004

In mammals, expiration is lengthened by mid‐expiratory lung inflation (Breuer‐Hering Expiratory reflex; BHE). The central pathway mediating the BHE is paucisynaptic, converging on neurones in the rostral ventrolateral medulla. An in vitro neonatal rat brainstem–lung preparation in which mid‐expiratory inflation lengthens expiration was used to study afferent modulation of respiratory neurone activity. Recordings were made from respiratory neurones in or near the pre‐Bötzinger Complex (preBötC). Respiratory neurone membrane properties and BHE‐induced changes in activity were characterized. Our findings suggest the following mechanisms for the BHE: (i) lung afferent signals strongly excite biphasic neurones that convey these signals to respiratory neurones in ventrolateral medulla; (ii) expiratory lengthening is mediated by inhibition of rhythmogenic and (pre)motoneuronal networks; and (iii) pre‐inspiratory (Pre‐I) neurones, some of which project to abdominal expiratory motoneurones, are excited during the BHE. These findings are qualitatively similar to studies of the BHE in vivo. Where there are differences, they can largely be accounted for by developmental changes and experimental conditions.
Shape memory alloy actuators in smart structures: Modeling and simulation
Authors: Seelecke Stefan , Mu¨ller Ingo
Published: February 2004

This review article gives an overview of the new and quickly developing field of shape memory alloy (SMA) actuators in smart structures. The focus is on the aspects of modeling and simulation of such structures, a task that goes beyond classical modeling approaches as it has to combine constitutive modeling with structural and control aspects in a highly interdisciplinary way. We review developments in each of these fields, trying to combine them into a smooth picture of how to treat the problem efficiently. After a discussion of modeling aspects with particular regard to actuator applications, the simulation of standard feedback control methods is demonstrated. Subsequently, model based methods from optimal control theory are presented, accounting for the strongly nonlinear and hysteretic material behavior of SMAs. Real-time optimal control methods are introduced and, finally, aspects of finite element implementation of an SMA actuator model are discussed and illustrated by the simulation of an adaptive aircraft wing. This review article cites 155 references.
Structure of the Uncleaved Human H1 Hemagglutinin from the Extinct 1918 Influenza Virus
Authors: Stevens James , Corper Adam L. , Basler Christopher F. , Taubenberger Jeffery K. , Palese Peter , Wilson Ian A.
Published: February 2004

The 1918 “Spanish” influenza pandemic represents the largest recorded outbreak of any infectious disease. The crystal structure of the uncleaved precursor of the major surface antigen of the extinct 1918 virus was determined at 3.0 angstrom resolution after reassembly of the hemagglutinin gene from viral RNA fragments preserved in 1918 formalin-fixed lung tissues. A narrow avian-like receptor-binding site, two previously unobserved histidine patches, and a less exposed surface loop at the cleavage site that activates viral membrane fusion reveal structural features primarily found in avian viruses, which may have contributed to the extraordinarily high infectivity and mortality rates observed during 1918.
Response of the
Saccharomyces cerevisiae
Mpk1 Mitogen-Activated Protein Kinase Pathway to Increases in Internal Turgor Pressure Caused by Loss of Ppz Protein Phosphatases
Authors: Merchan Stephanie , Bernal Dolores , Serrano Ramón , Yenush Lynne
Published: February 2004

The Mpk1 pathway of
Saccharomyces cerevisiae
is a key determinant of cell wall integrity. A genetic link between the Mpk1 kinase and the Ppz phosphatases has been reported, but the nature of this connection was unclear. Recently, the Ppz phosphatases were shown to be regulators of K
+
and pH homeostasis. Here, we demonstrate that Ppz-deficient strains display increased steady-state K
+
levels and sensitivity to increased KCl concentrations. Given these observations and the fact that K
+
is the major determinant of intracellular turgor pressure, we reasoned that the connection between
PPZ1
and -
2
and
MPK1
was due to the combination of increased internal turgor pressure in Ppz-deficient strains and cell wall instability observed in strains lacking
MPK1
. Accordingly, the
MPK1
gene was up-regulated, the Mpk1 protein was overexpressed, and the phosphorylated active form was more abundant in Ppz-deficient strains. Moreover, the expression of genes previously identified as targets of the Mpk1 pathway are also up-regulated in strains lacking
PPZ1
and -
2
. The transcriptional and posttranslational modifications of Mpk1 were not observed when the internal K
+
concentration (and thus turgor pressure) was lowered by disrupting the
TRK1
and -
2
K
+
transporter genes or when the cell wall was stabilized by the addition of sorbitol. Moreover, we present genetic evidence showing that both the Wsc1 and Mid2 branches of the Mpk1 pathway contribute to this response. Finally, despite its role in G
1
/S transition, increased levels of activated Mpk1 do not appear to be responsible for the cell cycle phenotype observed in Ppz-deficient strains.
The Ras/Protein Kinase A Pathway Acts in Parallel with the Mob2/Cbk1 Pathway To Effect Cell Cycle Progression and Proper Bud Site Selection
Authors: Schneper Lisa , Krauss Alicia , Miyamoto Ryan , Fang Shirley , Broach James R.
Published: February 2004

In
Saccharomyces cerevisiae
, Ras proteins connect nutrient availability to cell growth through regulation of protein kinase A (PKA) activity. Ras proteins also have PKA-independent functions in mitosis and actin repolarization. We have found that mutations in
MOB2
or
CBK1
confer a slow-growth phenotype in a
ras2
Δ background. The slow-growth phenotype of
mob2
Δ
ras2
Δ cells results from a G
1
delay that is accompanied by an increase in size, suggesting a G
1
/S role for Ras not previously described. In addition,
mob2
Δ strains have imprecise bud site selection, a defect exacerbated by deletion of
RAS2
. Mob2 and Cbk1 act to properly localize Ace2, a transcription factor that directs daughter cell-specific transcription of several genes. The growth and budding phenotypes of the double-deletion strains are Ace2 independent but are suppressed by overexpression of the PKA catalytic subunit, Tpk1. From these observations, we conclude that the PKA pathway and Mob2/Cbk1 act in parallel to determine bud site selection and promote cell cycle progression.
Candida albicans
Csy1p Is a Nutrient Sensor Important for Activation of Amino Acid Uptake and Hyphal Morphogenesis
Authors: Brega Elisa , Zufferey Rachel , Mamoun Choukri Ben
Published: February 2004

Candida albicans
is an important human pathogen that displays a remarkable ability to detect changes in its environment and to respond appropriately by changing its cell morphology and physiology. Serum- and amino acid-based media are known to induce filamentous growth in this organism. However, the mechanism by which amino acids induce filamentation is not yet known. Here, we describe the identification and characterization of the primary amino acid sensor of
C. albicans
, Csy1. We show that Csy1p plays an important role in amino acid sensing and filamentation. Loss of Csy1p results in a lack of amino acid-mediated activation of amino acid transport and a lack of induction of transcription of specific amino acid permease genes. Furthermore, a
csy1
Δ/
csy1
Δ strain, lacking Csy1p, is defective in filamentation and displays altered colony morphology in serum- and amino acid-based media. These data provide the first evidence that
C. albicans
utilizes the amino acid sensor Csy1p to probe its environment, coordinate its nutritional requirements, and determine its morphological state.
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits Have Divergent Roles in Virulence Factor Production in Two Varieties of the Fungal Pathogen
Cryptococcus neoformans
Authors: Hicks Julie K. , D'Souza Cletus A. , Cox Gary M. , Heitman Joseph
Published: February 2004

Our earlier findings established that cyclic AMP-dependent protein kinase functions in a signaling cascade that regulates mating and virulence of
Cryptococcus neoformans
var.
grubii
(serotype A). Mutants lacking the serotype A protein kinase A (PKA) catalytic subunit Pka1 are unable to mate, fail to produce melanin or capsule, and are avirulent in animal models, whereas mutants lacking the PKA regulatory subunit Pkr1 overproduce capsule and are hypervirulent. Because other mutations have been observed to confer different phenotypes in two diverged varieties of
C. neoformans
(
grubii
variety [serotype A] and
neoformans
variety [serotype D]), we analyzed the functions of the PKA genes in the serotype D
neoformans
variety. Surprisingly, the Pka1 catalytic subunit was not required for mating, haploid fruiting, or melanin or capsule production of serotype D strains. Here we identify a second PKA catalytic subunit gene,
PKA2
, that is present in both serotype A and D strains of
C. neoformans
. The divergent Pka2 catalytic subunit was found to regulate mating, haploid fruiting, and virulence factor production in serotype D strains. In contrast, Pka2 has no role in mating, melanin production, or capsule formation in serotype A strains. Our studies illustrate how different components of signaling pathways can be co-opted and functionally specialized during the evolution of related but distinct varieties or subspecies of a human fungal pathogen.
Chromatin Rearrangements in the
prnD-prnB
Bidirectional Promoter: Dependence on Transcription Factors
Authors: García Irene , Gonzalez Ramón , Gómez Dennis , Scazzocchio Claudio
Published: February 2004

The
prnD-prnB
intergenic region regulates the divergent transcription of the genes encoding proline oxidase and the major proline transporter. Eight nucleosomes are positioned in this region. Upon induction, the positioning of these nucleosomes is lost. This process depends on the specific transcriptional activator PrnA but not on the general GATA factor AreA. Induction of
prnB
but not
prnD
can be elicited by amino acid starvation. A specific nucleosomal pattern in the
prnB
proximal region is associated with this process. Under conditions of induction by proline, metabolite repression depends on the presence of both repressing carbon (glucose) and nitrogen (ammonium) sources. Under these repressing conditions, partial nucleosomal positioning is observed. This depends on the CreA repressor's binding to two specific
cis
-acting sites. Three conditions (induction by the defective PrnA80 protein, induction by amino acid starvation, and induction in the presence of an activated CreA) result in similar low transcriptional activation. Each results in a different nucleosome pattern, which argues strongly for a specific effect of each signal on nucleosome positioning. Experiments with trichostatin A suggest that both default nucleosome positioning and partial positioning under induced-repressed conditions depend on deacetylated histones.
A Non-Long Terminal Repeat Retrotransposon Family Is Restricted to the Germ Line Micronucleus of the Ciliated Protozoan
Tetrahymena thermophila
Authors: Fillingham Jeffrey S. , Thing Trine A. , Vythilingum Nama , Keuroghlian Alex , Bruno Deanna , Golding G. Brian , Pearlman Ronald E.
Published: February 2004

The ciliated protozoan
Tetrahymena thermophila
undergoes extensive programmed DNA rearrangements during the development of a somatic macronucleus from the germ line micronucleus in its sexual cycle. To investigate the relationship between programmed DNA rearrangements and transposable elements, we identified several members of a family of non-long terminal repeat (LTR) retrotransposons (retroposons) in
T. thermophila
, the first characterized in the ciliated protozoa. This multiple-copy retrotransposon family is restricted to the micronucleus of
T. thermophila
. The REP (
Tetrahymena
non-LTR retroposon) elements encode an ORF2 typical of non-LTR elements that contains apurinic/apyrimidinic endonuclease (APE) and reverse transcriptase (RT) domains. Phylogenetic analysis of the RT and APE domains indicates that the element forms a deep-branching clade within the non-LTR retrotransposon family. Northern analysis with a probe to the conserved RT domain indicates that transcripts from the element are small and heterogeneous in length during early macronuclear development. The presence of a repeated transposable element in the genome is consistent with the model that programmed DNA deletion in
T. thermophila
evolved as a method of eliminating deleterious transposons from the somatic macronucleus.
An
Entamoeba histolytica
LINE/SINE Pair Inserts at Common Target Sites Cleaved by the Restriction Enzyme-Like LINE-Encoded Endonuclease
Authors: Mandal Prabhat K. , Bagchi Anindya , Bhattacharya Alok , Bhattacharya Sudha
Published: February 2004

The non-long-terminal-repeat (non-LTR) retrotransposons (also called long interspersed repetitive elements [LINEs]) are among the oldest retroelements. Here we describe the properties of such an element from a primitive protozoan parasite,
Entamoeba histolytica
, that infects the human gut. This 4.8-kb element, called EhLINE1, is present in about 140 copies dispersed throughout the genome. The element belongs to the R4 clade of non-LTR elements. It has a centrally located reverse transcriptase domain and a restriction enzyme-like endonuclease (EN) domain at the carboxy terminus. We have cloned and expressed a 794-bp fragment containing the EN domain in
Escherichia coli.
The purified protein could nick supercoiled pBluescript DNA to yield open circular and linear DNAs. The conserved PDX
12-14
D motif was required for activity. Genomic sequences flanking the sites of insertion of EhLINE1 and the putative partner short interspersed repetitive element (SINE), EhSINE1, were analyzed. Both elements resulted in short target site duplications (TSD) upon insertion. A common feature was the presence of a short T-rich stretch just upstream of the TSD in most insertion sites. By sequence analysis an empty target site in the
E. histolytica
genome, known to be occupied by EhSINE1, was identified. When a 176-bp fragment containing the empty site was used as a substrate for EN, it was prominently nicked on the bottom strand at the precise point of insertion of EhSINE1, showing that this SINE could use the LINE-encoded endonuclease for its insertion. The nick on the bottom strand was toward the right of the TSD, which is uncommon. The lack of strict target site-specificity of the restriction enzyme-like EN encoded by EhLINE1 is also exceptional. A model for retrotransposition of EhLINE1/SINE1 is presented.
Genetic Evidence for Interaction between η- and β-Tubulins
Authors: Ruiz F. , Dupuis-Williams P. , Klotz C. , Forquignon F. , Bergdoll M. , Beisson J. , Koll F.
Published: February 2004

The thermosensitive allelic mutations
sm19-1
and
sm19-2
of
Paramecium tetraurelia
cause defective basal body duplication: growth at the nonpermissive temperature yields smaller and smaller cells with fewer and fewer basal bodies. Complementation cloning of the
SM19
gene identified a new tubulin, eta-tubulin, showing low homology with each of the other five tubulins, α to ε, characterized in
P. tetraurelia
. In order to analyze η-tubulin functions, we used a genetic approach to identify interacting molecules. Among a series of extragenic suppressors of the
sm19-1
mutation, the
su3-1
mutation was characterized as an E288K substitution in the
β-PT2
gene coding for a β-tubulin, while the mutation

noc
r

1
conferring nocodazole resistance and localized in another β-tubulin gene,
β-PT3
, was shown to enhance the mutant phenotype. The interaction between η-tubulin and microtubules, revealed by genetic data, is supported by two further types of evidence: first, the mutant phenotype is rescued by taxol, which stabilizes microtubules; second, molecular modeling suggests that η-tubulin, like γ- and δ-tubulins, might be a microtubule minus-end capping molecule. The likely function of η-tubulin as part of a complex specifically involved in basal body biogenesis is discussed.
A WD40 Repeat Protein Regulates Fungal Cell Differentiation and Can Be Replaced Functionally by the Mammalian Homologue Striatin
Authors: Pöggeler Stefanie , Kück Ulrich
Published: February 2004

Fruiting body development in fungi is a complex cellular differentiation process that is controlled by more than 100 developmental genes. Mutants of the filamentous fungus
Sordaria macrospora
showing defects in fruiting body formation are pertinent sources for the identification of components of this multicellular differentiation process. Here we show that the sterile mutant pro11 carries a defect in the
pro11
gene encoding a multimodular WD40 repeat protein. Complementation analysis indicates that the wild-type gene or C-terminally truncated versions of the wild-type protein are able to restore the fertile phenotype in mutant pro11. PRO11 shows significant homology to several vertebrate WD40 proteins, such as striatin and zinedin, which seem to be involved in Ca
2+
-dependent signaling in cells of the central nervous system and are supposed to function as scaffolding proteins linking signaling and eukaryotic endocytosis. Cloning of a mouse cDNA encoding striatin allowed functional substitution of the wild-type protein with restoration of fertility in mutant pro11. Our data strongly suggest that an evolutionarily conserved cellular process controlling eukaryotic cell differentiation may regulate fruiting body formation.
ADAM Family Protein Mde10 Is Essential for Development of Spore Envelopes in the Fission Yeast
Schizosaccharomyces pombe
Authors: Nakamura Tomohiro , Abe Hiroko , Hirata Aiko , Shimoda Chikashi
Published: February 2004

We report the identification of
Schizosaccharomyces pombe mde10
+
as a gene possessing a FLEX element, which forms a binding site for the meiosis-specific transcription factor Mei4. In fact,
mde10
+
is transcribed only in diploid cells that are induced to meiosis in a Mei4-dependent manner. Western blot analysis indicated that the epitope-tagged Mde10 protein accumulates transiently during meiosis and then rapidly decreases. Mde10 is a multidomain protein containing a metalloprotease catalytic domain, a disintegrin domain, a cysteine-rich domain, and membrane-spanning regions, all of which are shared by members of the mammalian ADAM family. A fusion protein of Mde10 and green fluorescent protein localized to the endoplasmic reticulum during meiosis and was located at the peripheral region of spores at the end of meiosis. An
mde10
Δ deletion mutant showed no apparent defects in meiosis, sporulation, or spore germination. However, the mutant spores exhibited an aberrant surface appearance, in which the ragged outer spore wall was lost to a large extent. Furthermore,
mde10
Δ spores were found to be less tolerant to ethanol and diethyl ether than were wild-type spores. The mutagenic replacement of the conserved glutamic acid in the putative protease active site with an alanine residue did not affect the surface morphology or the resistance of spores to environmental stress. Our observations indicate that Mde10 is important in the development of the spore envelope, although this function of Mde10 seems to be independent of its metalloprotease activity.
The Ca
2+
/Calcineurin-Regulated
cup
Gene Family in
Dictyostelium discoideum
and Its Possible Involvement in Development
Authors: Coukell Barrie , Li Yi , Moniakis John , Cameron Anne
Published: February 2004

Changes in free intracellular Ca
2+
are thought to regulate several major processes during
Dictyostelium
development, including cell aggregation and cell type-specific gene expression, but the mechanisms involved are unclear. To learn more about Ca
2+
signaling and Ca
2+
homeostasis in this organism, we used suppression subtractive hybridization to identify genes up-regulated by high extracellular Ca
2+
. Unexpectedly, many of the genes identified belong to a novel gene family (termed
cup
) with seven members. In vegetative cells, the
cup
genes were up-regulated by high Ca
2+
but not by other ions or by heat, oxidative, or osmotic stress.
cup
induction by Ca
2+
was blocked completely by inhibitors of calcineurin and protein synthesis. In developing cells,
cup
expression was high during aggregation and late development but low during the slug stage. This pattern correlates closely with reported levels of free intracellular Ca
2+
during development. The
cup
gene products are highly homologous, acidic proteins possessing putative ricin domains. BLAST searches failed to reveal homologs in other organisms, but Western analyses suggested that Cup-like proteins might exist in certain other cellular slime mold species. Localization experiments indicated that Cup proteins are primarily cytoplasmic but become cell membrane-associated during Ca
2+
stress and cell aggregation. When
cup
expression was down-regulated by antisense RNA, the cells failed to aggregate. However, this developmental block was overcome by partially up-regulating
cup
expression. Together, these results suggest that the Cup proteins in
Dictyostelium
might play an important role in stabilizing and/or regulating the cell membrane during Ca
2+
stress and/or certain stages of development.
Flagellar Radial Spoke Protein 2 Is a Calmodulin Binding Protein Required for Motility in
Chlamydomonas reinhardtii
Authors: Yang Pinfen , Yang Chun , Sale Winfield S.
Published: February 2004

Genetic and morphological studies have revealed that the radial spokes regulate ciliary and flagellar bending. Functional and biochemical analysis and the discovery of calmodulin in the radial spokes suggest that the regulatory mechanism involves control of axonemal protein phosphorylation and calcium binding to spoke proteins. To identify potential regulatory proteins in the radial spoke, in-gel kinase assays were performed on isolated axonemes and radial spoke fractions. The results indicated that radial spoke protein 2 (RSP2) can bind ATP and transfer phosphate in vitro.
RSP2
was cloned and mapped to the
PF24
locus, a gene required for motility. Sequencing revealed that
pf24
contains a point mutation converting the first ATG to ATA, resulting in only trace amounts of RSP2 and confirming the
RSP2
mapping. Surprisingly, the sequence does not include signature domains for conventional kinases, indicating that RSP2 may not perform as a protein kinase in vivo. However, the predicted RSP2 protein sequence contains Ca
2+
-dependent calmodulin binding motifs and a GAF domain, a domain found in diverse signaling proteins for binding small ligands including cyclic nucleotides. As predicted from the sequence, recombinant RSP2 binds calmodulin in a calcium-dependent manner. We postulate that RSP2 is a regulatory subunit of the radial spoke involved in localization of calmodulin for control of motility.
Potential research participants’ views regarding researcher and institutional financial conflicts of interest
Authors: Kim S Y H , Millard R W , Nisbet P , Cox C , Caine E D
Published: February 2004

Background:Financial conflict of interest in clinical research is an area of active debate. While data exist on the perspectives and roles of academic institutions, investigators, industry sponsors, and scientific journals, little is known about the perspectives of potential research participants.

Methods:The authors surveyed potential research participants over the internet, using the Harris Interactive Chronic Illness Database. A potential research participant was defined by: (1) self report of diagnosis by a health care professional and (2) willingness to participate in clinical trials. Email invitations were sent to 20 205 persons with coronary artery disease, breast cancer, or depression; a total of 6363 persons were screened; of these, 86% or 5478 met inclusion criteria and completed the survey. The outcome measures were respondents’ ratings on: importance of knowing conflict of interest information, whether its disclosure ought to be required, and its effect on willingness to participate—across seven widely discussed scenarios of financial conflicts of interest (ranging from commercial funding to equity ownership).

Results:Majority responded that knowing conflict of interest information was “extremely” or “very” important; a larger majority felt financial conflicts of interest should be disclosed as part of informed consent (64% to 87%). In all seven scenarios, a majority was still willing to participate but in some scenarios a sizable minority would be wary of participation. Respondents were more wary of individual than institutional conflicts of interest. Illness group and sociodemographic factors had modest effects and did not affect the main trends.

Conclusions:The prevailing practice of non-disclosure of financial conflicts of interest in clinical research appears contrary to the values of potential research participants.
Proceeding with clinical trials of animal to human organ transplantation: a way out of the dilemma
Authors: Ravelingien A , Mortier F , Mortier E , Kerremans I , Braeckman J
Published: February 2004

The transplantation of porcine organs to humans could in the future be a solution to the worldwide organ shortage, but is to date still highly experimental. Further research on the potential effects of crossing the species barrier is essential before clinical application is acceptable. However, many crucial questions on efficacy and safety will ultimately only be answered by well designed and controlled solid organ xenotransplantation trials on humans. This paper is concerned with the question under which conditions, given the risks involved and the ethical issues raised, such clinical trials should be resumed. An alternative means of overcoming the safety and ethical issues is suggested: willed body donation for scientific research in the case of permanent vegetative status. This paper argues that conducting trials on such bodies with prior consent is preferable to the use of human subjects without lack of brain function.
Challenge studies of human volunteers: ethical issues
Authors: Hope T , McMillan J
Published: February 2004

There is a long history of medical research that involves intentionally infecting healthy people in order to study diseases and their treatments. Such research—what might be called “human challenge studies”—are an important strand of much current research—for example, in the development of vaccinations. The many international and national guidelines about the proper conduct of medical research do not specifically address human challenge studies. In this paper we review the guidelines on the risk of harm that healthy volunteers may be exposed to in the course of medical research. We examine the ethical arguments that are implicit or explicit in these guidelines. We then ask whether there is reason for limiting such studies on grounds independent of risk of harm. We conclude that the major ethical concern with challenge studies is that of risk of harm and that the fact that a study is a challenge study is not a wrong in itself.
Public attitudes towards the use of primary care patient record data in medical research without consent: a qualitative study
Authors: Robling M R , Hood K , Houston H , Pill R , Fay J , Evans H M
Published: February 2004

Objectives:Recent legislative changes within the United Kingdom have stimulated professional debate about access to patient data within research. However, there is currently little awareness of public views about such research. The authors sought to explore attitudes of the public, and their lay representatives, towards the use of primary care medical record data for research when patient consent was not being sought.

Methods:49 members of the public and four non-medical members of local community health councils in South Wales, UK gave their views on the value and acceptability of three current research scenarios, each describing access to data without patient consent.

Results:Among focus group participants, awareness of research in primary care was low, and the appropriateness of general practitioners as researchers was questioned. There was general support for research but also concerns expressed about data collection without consent. These included lack of respect and patient control over the process. Unauthorised access to data by external agencies was a common fear. Current data collection practices, including population based disease registers elicited much anxiety. The key informants were equally critical of the scenarios and generally less accepting.

Conclusions:This exploratory study has highlighted a number of areas of public concern when medical records are accessed for research without patient consent. Public acceptability regarding the use of medical records in research cannot simply be assumed. Further work is required to determine how widespread such views are and to inform those advising on confidentiality issues.
Non-therapeutic research with minors: how do chairpersons of German research ethics committees decide?: Figure 1
Authors: Lenk C , Radenbach K , Dahl M , Wiesemann C
Published: February 2004

Objectives:Clinical trials in humans in Germany—as in many other countries—must be approved by local research ethics committees (RECs). The current study has been designed to document and evaluate decisions of chairpersons of RECs in the problematic field of non-therapeutic research with minors. The authors’ purpose was to examine whether non-therapeutic research was acceptable for chairpersons at all, and whether there was certainty on how to decide in research trials involving more than minimal risk.

Design:In a questionnaire, REC chairpersons had to evaluate five different scenarios with (in parts) non-therapeutic research. The scenarios described realistic potential research projects with minors, involving increasing levels of risk for the research participants. The chairpersons had to decide whether the respective projects should be approved.

Methods:A total of 49 German REC chairpersons were sent questionnaires; 29 questionnaires were returned. The main measurements were approval or rejection of research scenarios.

Results:Chairpersons of German RECs generally tend to accept non-therapeutic research with minors if the apparent risk for the participating children is low. If the risk is clearly higher than “minimal”, the chairpersons’ decisions differ widely.

Conclusion:The fact that there seem to be different attitudes of chairpersons to non-therapeutic research with minors is problematic from an ethical point of view. It suggests a general uncertainty about the standards of protection for minor research participants in Germany. Therefore, further ethical and legal regulation of non-therapeutic research with minors in Germany seems necessary.
Differential membrane potential and ion current responses to different types of shear stress in vascular endothelial cells
Authors: Lieu Deborah K. , Pappone Pamela A. , Barakat Abdul I.
Published: February 2004

Vascular endothelial cells (ECs) distinguish among and respond differently to different types of fluid mechanical shear stress. Elucidating the mechanisms governing this differential responsiveness is the key to understanding why early atherosclerotic lesions localize preferentially in arterial regions exposed to low and/or oscillatory flow. An early and very rapid endothelial response to flow is the activation of flow-sensitive K+ and Cl− channels that respectively hyperpolarize and depolarize the cell membrane and regulate several important endothelial responses to flow. We have used whole cell current- and voltage-clamp techniques to demonstrate that flow-sensitive hyperpolarizing and depolarizing currents respond differently to different types of shear stress in cultured bovine aortic ECs. A steady shear stress level of 10 dyn/cm2 activated both currents leading to rapid membrane hyperpolarization that was subsequently reversed to depolarization. In contrast, a steady shear stress of 1 dyn/cm2 only activated the hyperpolarizing current. A purely oscillatory shear stress of 0 ± 10 dyn/cm2 with an oscillation frequency of either 1 or 0.2 Hz activated the hyperpolarizing current but only minimally the depolarizing current, whereas a 5-Hz oscillation activated neither current. These results demonstrate for the first time that flow-activated ion currents exhibit different sensitivities to shear stress magnitude and oscillation frequency. We propose that flow-sensitive ion channels constitute components of an integrated mechanosensing system that, through the aggregate effect of ion channel activation on cell membrane potential, enables ECs to distinguish among different types of flow.