bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidemiology of Neural Tube Defects
Authors: Frey Lauren , Hauser W. Allen
Published: June 2003

Summary: Neural tube defects (NTDs)—malformations secondary to abnormal neural tube closure between the third and fourth weeks of gestational age—have a complex and imperfectly understood etiology in which both genetic and environmental factors appear to be involved. A number of specific chromosomal or single‐gene disorders, presumably not affected by environmental influences, are associated with the development of NTDs, but such syndromal cases account for a small proportion of NTDs in live‐born infants. Analysis of recurrence patterns within families and of twin‐concordance data provides evidence of a genetic influence in nonsyndromal cases, but factors such as socioeconomic status and geographic area (independent of race or ethnicity) are also associated with variations in the incidence of NTDs. The prevalence at birth of both anencephaly and spina bifida has decreased, but the advent of antenatal diagnosis and elective termination of affected pregnancies has undermined the reliability of birth prevalence rate as an estimate of incidence. Some occupational and other exposures, including maternal use of antiepileptic drugs (AEDs), are associated with increased risk for NTDs. Among women who have had an NTD‐affected pregnancy, recurrence risk is markedly higher than the risk for a first NTD‐affected pregnancy in the general population. There is strong evidence, overall, for a protective effect of adequate folate consumption. In some high‐risk groups, however, such as women taking AEDs, folate supplementation has not been proven to reduce NTD risk.
Role of the Hepatocyte Nuclear Factor 4α in Control of the Pregnane X Receptor During Fetal Liver Development
Authors: Kamiya Akihide , Inoue Yusuke , Gonzalez Frank J.
Published: June 2003

The fetal liver, the major site of hematopoiesis during embryonic development, acquires additional functions near birth. Among the important liver functions is the response to xenobiotic exposure due to expression of several cytochromes P450 (CYP) and drug efflux transporters. Expression of these genes is regulated by nuclear receptors such as the pregnane X receptor (PXR). In this study, regulation of xenobiotic responses during fetal liver development was analyzed using a fetal hepatocyte primary culture system derived from embryonic day 15 (E15) livers. Hepatocyte nuclear factor (HNF) 4α regulates the expression of many genes preferentially in the liver. Expression of several xenobiotic response genes as well as HNF4α was increased in fetal hepatocytes stimulated by the hepatic maturation factors oncostatin M (OSM) and Matrigel. To determine the contribution of HNF4α to xenobiotic responses in the fetal liver, fetal hepatocytes containing floxed HNF4α alleles were cultured and the HNF4α gene was inactivated by infection with an adenovirus containing the Cre gene. Expression of CYP3A11 and PXR was suppressed by inactivation of HNF4α. An HNF4α binding site was characterized in the PXR promoter and found to be required for activation of the PXR promoter in fetal hepatocytes. In conclusion, HNF4α is the key transcription factor regulating responses to xenobiotics through activation of the PXR gene during fetal liver development.
BMPs signal alternately through a SMAD or FRAP–STAT pathway to regulate fate choice in CNS stem cells
Authors: Rajan Prithi , Panchision David M. , Newell Laura F. , McKay Ronald D.G.
Published: June 2003

The ability of stem cells to generate distinct fates is critical for the generation of cellular diversity during development. Central nervous system (CNS) stem cells respond to bone morphogenetic protein (BMP) 4 by differentiating into a wide variety of dorsal CNS and neural crest cell types. We show that distinct mechanisms are responsible for the generation of two of these cell types, smooth muscle and glia. Smooth muscle differentiation requires BMP-mediated Smad1/5/8 activation and predominates where local cell density is low. In contrast, glial differentiation predominates at high local densities in response to BMP4 and is specifically blocked by a dominant-negative mutant Stat3. Upon BMP4 treatment, the serine-threonine kinase FKBP12/rapamycin-associated protein (FRAP), mammalian target of rapamycin (mTOR), associates with Stat3 and facilitates STAT activation. Inhibition of FRAP prevents STAT activation and glial differentiation. Thus, glial differentiation by BMP4 occurs by a novel pathway mediated by FRAP and STAT proteins. These results suggest that a single ligand can regulate cell fate by activating distinct cytoplasmic signals.
The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin
Authors: Kovar David R. , Kuhn Jeffrey R. , Tichy Andrea L. , Pollard Thomas D.
Published: June 2003

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.
The COOH-terminal domain of agrin signals via a synaptic receptor in central nervous system neurons
Authors: Hoover Cameron L. , Hilgenberg Lutz G.W. , Smith Martin A.
Published: June 2003

Agrin is a motor neuron–derived factor that directs formation of the postsynaptic apparatus of the neuromuscular junction. Agrin is also expressed in the brain, raising the possibility that it might serve a related function at neuron–neuron synapses. Previously, we identified an agrin signaling pathway in central nervous system (CNS) neurons, establishing the existence of a neural receptor that mediates responses to agrin. As a step toward identifying this agrin receptor, we have characterized the minimal domains in agrin that bind and activate it. Structures required for agrin signaling in CNS neurons are contained within a 20-kD COOH-terminal fragment of the protein. Agrin signaling is independent of alternative splicing at the z site, but requires sequences that flank it because their deletion results in a 15-kD fragment that acts as an agrin antagonist. Thus, distinct regions within agrin are responsible for receptor binding and activation. Using the minimal agrin fragments as affinity probes, we also studied the expression of the agrin receptor on CNS neurons. Our results show that both agrin and its receptor are concentrated at neuron–neuron synapses. These data support the hypothesis that agrin plays a role in formation and/or function of CNS synapses.
A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor–regulated channel
Authors: Iwata Yuko , Katanosaka Yuki , Arai Yuji , Komamura Kazuo , Miyatake Kunio , Shigekawa Munekazu
Published: June 2003

Disruption of the dystrophin–glycoprotein complex caused by genetic defects of dystrophin or sarcoglycans results in muscular dystrophy and/or cardiomyopathy in humans and animal models. However, the key early molecular events leading to myocyte degeneration remain elusive. Here, we observed that the growth factor–regulated channel (GRC), which belongs to the transient receptor potential channel family, is elevated in the sarcolemma of skeletal and/or cardiac muscle in dystrophic human patients and animal models deficient in dystrophin or δ-sarcoglycan. However, total cell GRC does not differ markedly between normal and dystrophic muscles. Analysis of the properties of myotubes prepared from δ-sarcoglycan–deficient BIO14.6 hamsters revealed that GRC is activated in response to myocyte stretch and is responsible for enhanced Ca2+ influx and resultant cell damage as measured by creatine phosphokinase efflux. We found that cell stretch increases GRC translocation to the sarcolemma, which requires entry of external Ca2+. Consistent with these findings, cardiac-specific expression of GRC in a transgenic mouse model produced cardiomyopathy due to Ca2+ overloading, with disease expression roughly parallel to sarcolemmal GRC levels. The results suggest that GRC is a key player in the pathogenesis of myocyte degeneration caused by dystrophin–glycoprotein complex disruption.
Regulation of leading edge microtubule and actin dynamics downstream of Rac1
Authors: Wittmann Torsten , Bokoch Gary M. , Waterman-Storer Clare M.
Published: June 2003

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, “pioneer” MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.
Recording architecture at the archaeological site of Uxmal, Mexico: A historical and contemporary view
Authors: Desmond Lawrence G. , Bryan Paul G.
Published: June 2003

Architecture at the Maya archaeological site of Uxmal in Yucatan, Mexico has been documented since the 17th century by historians, archaeologists, illustrators, photographers, and most recently photogrammetrists. This paper reviews the work of the major contributors to recording of architecture at Uxmal with a particular focus on the 19th century stereophotography of Alice and Augustus Le Plongeon who worked to record all the standing architecture at Uxmal, and the close range stereophotogrammetric projects that recorded the Adivino Pyramid during the late 20th century.
Practical experiences with automatic aerial triangulation using different software packages
Authors: Büyüksali̇h Gürcan , Li Zhang
Published: June 2003

Digital photogrammetry and softcopy workstations have added a new dimension to aerial triangulation, and some of the previously manual procedures are now available in automated form. Also, commercial software packages for automatic aerial triangulation came on to the market in the last decade. In this study, the results of automatic aerial triangulation using four triangulation software packages, Z/I Imaging's MATCH‐AT (ISMAT) and ISAT, LH Systems’ MST and VirtuoZo AAT, are presented. For the comparison of the results of the tie point extraction and the bundle block adjustment for triangulation, a sub‐block from the Uster block in Switzerland was selected and used. Differences between these software packages are discussed with respect to their functionality and performance in detail. The achieved standard deviation of image coordinates varies between 0·2 and 0·5 pixel for the systems tested. The more signalised control points and check points to be used, the better the σ0 estimate which could be expected. The average number of extracted tie points varied from system to system with respect to the matching method and the resolution of the images used in the programs. The digital aerial triangulation of the Uster sub‐block was faster than analytical triangulation and the elapsed time for triangulation of one image using different systems varies between about 0·5 and 6 min. Nevertheless, results also show that automated aerial triangulation is still prone to error in all systems. Image matching produces many blunders, which can only be handled efficiently by using a sophisticated blunder detection procedure, as is available with robust estimation. This is not the case for all the systems tested, however. There still remains a lot to be done in terms of development (improvement of matching, for example, by integrating robust estimation for error detection at an early stage, implementation of on‐line triangulation, better internal quality control procedures, more transparent and simple user interfaces and more useful information for the user with better software manuals). However, it was shown that automatic aerial triangulation provides higher redundancy and reliability of results and much more economical photogrammetric practice than ever before.
The Anatomy of Loneliness
Authors: Cacioppo John T. , Hawkley Louise C. , Berntson Gary G.
Published: June 2003

Loneliness is a potent but little understood risk factor for broad-based morbidity and mortality. We review five social neurobehavioral mechanisms that may account for this association. The evidence suggests that different mechanisms explain short-term and long-term effects, and that the long-term effects operate through multiple pathways. Implications for the design of interventions are discussed.
Loneliness and Peer Relations in Childhood
Authors: Asher Steven R. , Paquette Julie A.
Published: June 2003

Although loneliness is a normative experience, there is reason to be concerned about children who are chronically lonely in school. Research indicates that children have a fundamental understanding of what it means to be lonely, and that loneliness can be reliably measured in children. Most of the research on loneliness in children has focused on the contributions of children's peer relations to their feelings of well-being at school. Loneliness in children is influenced by how well accepted they are by peers, whether they are overtly victimized, whether they have friends, and the durability and quality of their best friendships. Findings from this emerging area of research provide a differentiated picture of how children's peer experiences come to influence their emotional well-being.
Restraining the Genuine Homo Economicus: Why the Economy Cannot Be Divorced from Its Governance
Authors: Skaperdas Stergios
Published: June 2003

The Homo economicus of traditional economics is far from being completely self‐interested, rational, or as individualistic as he is purported to be; he will haggle to death over price but will not take what he wants by force. Implicitly, he is assumed to behave ruthlessly within a well‐defined bubble of sainthood. Based on a simple model, I first examine what occurs when this assumption is relaxed and genuine, amoral Homo economici interact. Productivity can be inversely related to compensation; a longer shadow of the future can intensify conflict; and more competition among providers of protection reduces welfare. The patently inefficient outcomes that follow call for restraining self‐interest, for finding ways to govern markets. I then review some of the different ways of creating restraints, from the traditional social contract, to the hierarchical domination of kings and lords, to modern forms of governance. Checks and balances, wider representation, the bureaucratic form of organization, and other ingredients of modern governance can partly be thought of as providing restraints to the dark side of self‐interest. Though highly imperfect, these restraints are better than the alternative, which typically involves autocratic, amateurish, and corrupt rule. Then, thinking of most problems in terms of a first‐best economic model is practically and scientifically misguided.
Politics as Tragedy in Several Acts
Authors: Buchanan James M.
Published: June 2003

James Madison noted that government would be unnecessary if men were angels. The shortfall of value between the idealized anarchy of a society of angels and the society of men as they are measures the tragedy of politics, which emerges at several levels. Resources are required for the maintenance of order, and those agents who are guardians of order will not, themselves, be angels. Further, rents promised to such agents will attract efforts at capture. Reductions in the measure of the tragedy remain possible through both shifts in ethical standards, at all levels, and shifts in institutional‐constitutional structures.
Asynchronous Cell Cycle and Asymmetric Vacuolar Inheritance in True Hyphae of
Candida albicans
Authors: Barelle Caroline J. , Bohula Erin A. , Kron Stephen J. , Wessels Deborah , Soll David R. , Schäfer Annette , Brown Alistair J. P. , Gow Neil A. R.
Published: June 2003

Candida albicans
forms unconstricted hyphae in serum-containing medium that are divided into discrete compartments. Time-lapse photomicroscopy, flow cytometry, and a novel three-dimensional imaging system were used to demonstrate that the kinetics and cell cycle events accompanying hyphal development were correlated with dynamic changes in vacuole morphology and the pattern of vacuole inheritance. Apical cells of hyphae underwent continuous extension before and after the first cytokinesis event. However, the resulting mother cell and sub-apical compartments did not immediately reenter the cell cycle and instead underwent cell cycle arrest before reentering the cycle. Vacuole was inherited asymmetrically at cytokinesis so that the distal, arrested compartments inherited most vacuole and the growing apical cell inherited most cytoplasm. Hydroxyurea release experiments demonstrated that the arrested, vacuolated hyphal compartments were in the G
1
phase of the cycle. The period of cell cycle arrest was decreased by the provision of assimilatable forms of nitrogen, suggesting that the hyphal cell cycle is regulated by nitrogen limitation that results in sup-apical cell cycle arrest. This pattern of growth is distinct from that of the synchronous, symmetrical development of pseudohyphae of
C. albicans
and other yeast species. These observations suggest that the cellular vacuole space correlates with alterations in the cell cycles of different cell types and that the total organelle space may influence size-regulated functions and hence the timing of the eukaryotic cell cycle.
Candida albicans VPS11
Is Required for Vacuole Biogenesis and Germ Tube Formation
Authors: Palmer Glen E. , Cashmore Annette , Sturtevant Joy
Published: June 2003

The
Candida albicans
vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C
vps
(vacuolar protein sorting) mutants of
Saccharomyces cerevisiae
are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study
C. albicans
homologues of the
S. cerevisiae
class C
VPS
genes have been identified. Deletion of a
C. albicans VPS11
homologue resulted in a number of phenotypes that closely resemble those of the class C
vps
mutants of
S. cerevisiae
, including the absence of a vacuolar compartment. The
C. albicans vps11Δ
mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore,
vps11Δ
strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.
A Gene Expressed during Sexual and Asexual Sporulation in
Phytophthora infestans
Is a Member of the Puf Family of Translational Regulators
Authors: Cvitanich Cristina , Judelson Howard S.
Published: June 2003

A gene from
Phytophthora infestans
that was previously identified as being induced during the development of sexual spores was also found to be active during asexual sporulation. The gene,
M90
, was expressed as a 3.1-kb primary transcript containing two introns and was predicted to encode a member of the Puf family of translational regulators. The protein showed up to 51% amino acid identity to other Puf proteins within its 353-amino-acid RNA-binding domain. Little similarity extended beyond this region, as noted for other members of the family. Expression of
M90
was measured by using RNA blots and transformants of
P. infestans
expressing a fusion between the
M90
promoter and the β-glucuronidase (GUS) gene. A 1.3-kb promoter fragment conferred the normal
M90
pattern of expression to the GUS reporter in transformants. In matings, expression was first detected in male and female gametangial initials and persisted in mature oospores. Expression was also observed in hyphal tips just prior to asexual sporulation, in sporangiophores, in mature sporangia, and in zoospores. The signal quickly disappeared once spores made the transition to hyphae after germination. Nutrient limitation did not induce the gene. Potential roles for a translational regulator during both sexual development and asexual sporulation are discussed.
Expression of Conventional and Unconventional Actins in
Chlamydomonas reinhardtii
upon Deflagellation and Sexual Adhesion
Authors: Hirono Masafumi , Uryu Satomi , Ohara Akio , Kato-Minoura Takako , Kamiya Ritsu
Published: June 2003

Chlamydomonas
has two actin genes, one coding for a conventional actin and the other coding for a highly divergent actin. The divergent actin NAP (for “novel actin-like protein”) is expressed only negligibly in wild-type cells but abundantly in a null mutant of conventional actin, the
ida5
mutant. The presence of the dormant NAP gene suggests that NAP may also have its own function in wild-type cells under some conditions. However, no specific functions have been suggested. In this study, we examined the expression of actin and NAP in wild-type and
ida5
cells under conditions where actin function has been shown to be important. We found that deflagellation induces the expression of NAP as well as that of actin in wild-type cells. The expressed NAP becomes localized to the regrown flagella, apparently without being associated with dynein. Mating of gametes also increased the expression of actin in wild-type cells and that of NAP in
ida5
cells, resulting in accumulation of these proteins in flagella (in both wild-type and
ida5
cells) and the fertilization tubule (only in wild-type cells). However, it did not induce significant NAP expression in wild-type cells. These and other observations suggest that the expression of actin and NAP mRNAs is controlled by two discrete mechanisms and that NAP plays a role in flagellar formation in wild-type cells.
Pheromone-Induced G2Arrest in the Phytopathogenic FungusUstilago maydis
Authors: García-Muse Tatiana , Steinberg Gero , Pérez-Martín José
Published: June 2003

In the corn smut fungusUstilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response inU. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone,U. maydiscells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G2phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.
Scw1p Antagonizes the Septation Initiation Network To Regulate Septum Formation and Cell Separation in the Fission Yeast
Schizosaccharomyces pombe
Authors: Jin Quan-Wen , McCollum Dannel
Published: June 2003

Cytokinesis in the fission yeast
Schizosaccharomyces pombe
is regulated by a signaling pathway termed the septation initiation network (SIN). The SIN is essential for initiation of actomyosin ring constriction and septum formation. In a screen to search for mutations that can rescue the
sid2
-
250
SIN mutant, we obtained
scw1
-
18
. Both the
scw1
-
18
mutant and the
scw1
deletion mutant (
scw1
Δ mutant), have defects in cell separation. Both the
scw1
-
18
and
scw1
Δ mutations rescue the growth defects of not just the
sid2
-
250
mutant but also the other temperature-sensitive SIN mutants. Other cytokinesis mutants, such as those defective for actomyosin ring formation, are not rescued by
scw1
Δ.
scw1
Δ does not seem to rescue the SIN by restoring SIN signaling defects. However,
scw1
Δ may function downstream of the SIN to promote septum formation, since
scw1
Δ can rescue the septum formation defects of the
cps1
-
191
β-1,3-glucan synthase mutant, which is required for synthesis of the primary septum.
Novel Rho GTPase Involved in Cytokinesis and Cell Wall Integrity in the Fission Yeast
Schizosaccharomyces pombe
Authors: Santos Beatriz , Gutiérrez Javier , Calonge Teresa M. , Pérez Pilar
Published: June 2003

The Rho family of GTPases is present in all eukaryotic cells from yeast to mammals; they are regulators in signaling pathways that control actin organization and morphogenetic processes. In yeast, Rho GTPases are implicated in cell polarity processes and cell wall biosynthesis. It is known that Rho1 and Rho2 are key proteins in the construction of the cell wall, an essential structure that in
Schizosaccharomyces pombe
is composed of β-glucan, α-glucan, and mannoproteins. Rho1 regulates the synthesis of 1,3-β-
d
-glucan by activation of the 1,3-β-
d
-glucan synthase, and Rho2 regulates the synthesis of α-glucan by the 1,3-α-
d
-glucan synthase Mok1. Here we describe the characterization of another Rho GTPase in fission yeast, Rho4.
rho4
Δ cells are viable but display cell separation defects at high temperature. In agreement with this observation, Rho4 localizes to the septum. Overexpression of
rho4
+
causes lysis and morphological defects. Several lines of evidence indicate that both
rho4
+
deletion or
rho4
+
overexpression result in a defective cell wall, suggesting an additional role for Rho4 in cell wall integrity.
rho4
Δ cells also accumulate secretory vesicles around the septum and are defective in actin polarization. We propose that Rho4 could be involved in the regulation of the septum degradation during cytokinesis.
A
Saccharomyces cerevisiae
Mutant Unable To Convert Glucose to Glucose-6-Phosphate Accumulates Excessive Glucose in the Endoplasmic Reticulum due to Core Oligosaccharide Trimming
Authors: Miseta Attila , Tökés-Füzesi Margit , Aiello David P. , Bedwell David M.
Published: June 2003

d
-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast
Saccharomyces cerevisiae
has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu
3
Man
9
GlcNac
2
effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as
alg5Δ
or
alg6Δ
, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [
14
C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca
2+
uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.
Rapamycin Treatment Results in GATA Factor-Independent Hyperphosphorylation of the Proline Utilization Pathway Activator in
Saccharomyces cerevisiae
Authors: Saxena Deepti , Kannan K. B. , Brandriss Marjorie C.
Published: June 2003

Treatment of
Saccharomyces cerevisiae
cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation. Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes. The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin. Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment. Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p. The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene
PUT1
were also examined. We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced
PUT1
expression. Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased
PUT1
expression. Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation. A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.
RBP38, a Novel RNA-Binding Protein from Trypanosomatid Mitochondria, Modulates RNA Stability
Authors: Sbicego Sandro , Alfonzo Juan D. , Estévez Antonio M. , Rubio Mary Anne T. , Kang Xuedong , Turck Christoph W. , Peris Marian , Simpson Larry
Published: June 2003

We describe here the isolation and characterization of a novel RNA-binding protein, RBP38, from
Leishmania tarentolae
mitochondria. This protein does not contain any known RNA-binding motifs and is highly conserved among the trypanosomatids, but no homologues were found in other organisms. Recombinant LtRBP38 binds single and double-stranded (ds) RNA substrates with dissociation constants in the 100 nM range, as determined by fluorescence polarization analysis. Downregulation of expression of the homologous gene, TbRBP38, in procyclic
Trypanosoma brucei
by using conditional dsRNA interference resulted in 80% reduction of steady-state levels of RNAs transcribed from both maxicircle and minicircle DNA. In organello pulse-chase labeling experiments were used to determine the stability of RNAs in mitochondria that were depleted of TbRBP38. The half-life of metabolically labeled RNA decreased from ∼160 to ∼60 min after depletion. In contrast, there was no change in transcriptional activity. These observations suggest a role of RBP38 in stabilizing mitochondrial RNA.
Dyskinetoplastic
Trypanosoma brucei
Contains Functional Editing Complexes
Authors: Domingo Gonzalo J. , Palazzo Setareh S. , Wang Bingbing , Pannicucci Brian , Salavati Reza , Stuart Kenneth D.
Published: June 2003

Mitochondrial pre-mRNAs undergo posttranscriptional RNA editing as directed by small guide RNAs (gRNAs) to produce functional mRNAs in kinetoplastid protozoa. The pre-mRNAs and gRNAs are encoded in the maxicircle and minicircle components, respectively, of the kinetoplastid mitochondrial DNA (kDNA), and editing is catalyzed by a multienzyme protein complex.
Trypanosoma evansi
AnTat3/3, which lacks maxicircles but retains a single class of minicircles, and a dyskinetoplastic mutant of
Trypanosoma brucei
EATRO164, which is devoid of kDNA, were both shown to retain genes and proteins for the editing complex. The proteins are present in complexes that immunoprecipitate and sediment indistinguishably from wild-type complexes. The complexes catalyze precleaved insertion and deletion editing as well as full-round deletion editing in vitro. Thus, mutants which lack the natural substrates for RNA editing and all or most gRNAs retain editing complexes that contain the four primary catalytic activities of editing and function in editing, at least in vitro. Therefore neither pre-mRNA nor gRNA is required to form functional RNA-editing complexes.
TbMP44 Is Essential for RNA Editing and Structural Integrity of the Editosome inTrypanosoma brucei
Authors: Wang Bingbing , Ernst Nancy Lewis , Palazzo Setareh S. , Panigrahi Aswini K. , Salavati Reza , Stuart Kenneth
Published: June 2003

RNA editing produces mature mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridylates. It is catalyzed by a multiprotein complex, the editosome. We identified TbMP44 among the components of enriched editosomes by a combination of mass spectrometry and DNA sequence database analysis. Inactivation of an ectopic TbMP44 allele in cells in which the endogenous alleles were disrupted abolished RNA editing, inhibited cell growth, and was eventually lethal to bloodstream form trypanosomes. Loss of TbMP44 mRNA was followed initially by a reduction in the editosome sedimentation coefficient and then by the absence of other editosome proteins despite the presence of the mRNA. Reactivation ofTbMP44gene expression resulted in the resumption of cell growth and the reappearance of editosomes. These data indicate that TbMP44 is a component of the editosome that is essential for editing and critical for the structural integrity of the editosome.
Guanyl Nucleotide Exchange Factor Sql2 and Ras2 Regulate Filamentous Growth in
Ustilago maydis
Authors: Müller Philip , Katzenberger Jörg D. , Loubradou Gabriel , Kahmann Regine
Published: June 2003

The cyclic AMP (cAMP)-signaling pathway regulates cell morphology and plays a crucial role during pathogenic development of the plant-pathogenic fungus
Ustilago maydis
. Strains lacking components of this signaling pathway, such as the Gα-subunit Gpa3 or the adenylyl cyclase Uac1, are nonpathogenic and grow filamentously. On the other hand, strains exhibiting an activated cAMP pathway due to a dominant-active allele of
gpa3
display a glossy colony phenotype and are unable to proliferate in plant tumors. Here we present the identification of
sql2
as a suppressor of the glossy colony phenotype of a

gpa3
Q206L

strain.
sql2
encodes a protein with similarity to CDC25-like guanine nucleotide exchange factors, which are known to act on Ras proteins. Overexpression of
sql2
leads to filamentous growth that cannot be suppressed by exogenous cAMP, suggesting that Sql2 does not act upstream of Uac1. To gain more insight in signaling processes regulated by Sql2, we isolated two genes encoding Ras proteins. Expression of dominant active alleles of
ras1
and
ras2
showed that Ras2 induces filamentous growth while Ras1 does not affect cell morphology but elevates pheromone gene expression. These results indicate that Ras1 and Ras2 fulfill different functions in
U. maydis
. Moreover, observed similarities between the filaments induced by
sql2
and
ras2
suggest that Sql2 is an activator of Ras2. Interestingly,
sql2
deletion mutants are affected in pathogenic development but not in mating, indicating a specific function of
sql2
during pathogenesis.
Genetic Interactions of the E3 Ubiquitin Ligase Component FbxA with Cyclic AMP Metabolism and a Histidine Kinase Signaling Pathway during
Dictyostelium discoideum
Development
Authors: Tekinay Turgay , Ennis Herbert L. , Wu Mary Y. , Nelson Margaret , Kessin Richard H. , Ratner David I.
Published: June 2003

Dictyostelium discoideum
amoebae with an altered
fbxA
gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of
fbxA
and regulated proteolysis, we have recovered the promoter of
fbxA
and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it is also expressed in developing spore cells. Because
fbxA
cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene
regA
, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of
dhkA
, a gene encoding a two-component histidine kinase known to influence
Dictyostelium
development. RegA appears precociously and in greater amounts in the
fbxA
mutant than in the wild type, but in an
fbx
A/
dhk
A double mutant, RegA is restored to wild-type levels. Because the basis of
regA
suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined. The levels of cAMP are relatively constant during multicellular development in all strains except the
dhkA
mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant
dhkA/fbxA
is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both
fbxA
and
dhkA
genes act earlier than previously thought.
Changing Patterns of Gene Expression in
Dictyostelium
Prestalk Cell Subtypes Recognized by In Situ Hybridization with Genes from Microarray Analyses
Authors: Maeda Mineko , Sakamoto Haruyo , Iranfar Negin , Fuller Danny , Maruo Toshinari , Ogihara Satoshi , Morio Takahiro , Urushihara Hideko , Tanaka Yoshimasa , Loomis William F.
Published: June 2003

We used microarrays carrying most of the genes that are developmentally regulated in
Dictyostelium
to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in
Dictyostelium
and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.
MFE1, a Member of the Peroxisomal Hydroxyacyl Coenzyme A Dehydrogenase Family, Affects Fatty Acid Metabolism Necessary for Morphogenesis in
Dictyostelium
spp
Authors: Matsuoka Satomi , Saito Tamao , Kuwayama Hidekazu , Morita Naoki , Ochiai Hiroshi , Maeda Mineko
Published: June 2003

β-Oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or
d
-bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that
Dictyostelium
has a gene,
mfeA
, encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene,
mfeB
, encodes MFE2 with homology to the enoyl-CoA hydratase domain. When grown on a diet of bacteria,
Dictyostelium
cells in which
mfeA
is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids that remained throughout the developmental stages. Such a persistent accumulation was not detected in wild-type cells or axenically grown mutant cells. Bacterial phosphatidylethanolamine that contains abundant cyclopropane fatty acids inhibited the development of even axenically grown mutant cells, while dipalmitoyl phosphatidylethanolamine did not. These results suggest that MFE1 protects the cells from the increase of the harmful xenobiotic fatty acids incorporated from their diets and optimizes cellular lipid composition for proper development. Hence, we propose that this enzyme plays an irreplaceable role in the survival strategy of
Dictyostelium
cells to form spores for their efficient dispersal in nature.
Evidence that Synthesis of the
Saccharomyces cerevisiae
Mitochondrially Encoded Ribosomal Protein Var1p May Be Membrane Localized
Authors: Fiori Alessandro , Mason Thomas L. , Fox Thomas D.
Published: June 2003

The 5′-untranslated leaders of mitochondrial mRNAs appear to localize translation within the organelle.
VAR1
is the only yeast mitochondrial gene encoding a major soluble protein. A chimeric mRNA bearing the
VAR1
untranslated regions and the coding sequence for pre-Cox2p appears to be translated at the inner membrane surface. We propose that translation of the ribosomal protein Var1p is also likely to occur in close proximity to the inner membrane.
Commodification and exploitation: arguments in favour of compensated organ donation
Authors: de Castro L D
Published: June 2003

This paper takes the view that compensated donation and altruism are not incompatible. In particular, it holds that the arguments against giving compensation stand on weak rational grounds: (1) the charge that compensation fosters “commodification” has neither been specific enough to account for different types of monetary transactions nor sufficiently grounded in reality to be rationally convincing; (2) although altruism is commendable, organ donors should not be compelled to act purely on the basis of altruistic motivations, especially if there are good reasons to believe that significantly more lives can be saved and enhanced if incentives are put in place, and (3) offering compensation for organs does not necessarily lead to exploitation—on the contrary, it may be regarded as a necessity in efforts to minimise the level of exploitation that already exists in current organ procurement systems.
Presumed consent for transplantation: a dead issue after Alder Hey?
Authors: English V , Sommerville A
Published: June 2003

In the wake of scandals about the unauthorised retention of organs following postmortem examination, the issue of valid consent (or the lack of it) has returned to the forefront. Emphasis is put on obtaining explicit authorisation from the patient or family prior to any medical intervention, including those involving the dead. Although the controversies in the UK arose from the retention of human material for education or research rather than therapy, concern has been expressed that public mistrust could also adversely affect organ donation for transplantation. At the same time, however, the British Medical Association (BMA) continues to call for a shift to a system of presumed consent for organ transplantation. This apparent inconsistency can be justified because valid distinctions exist between the reasons requiring explicit consent for retention and the acceptability of presumed consent for transplantation. This paper argues for introducing a system of presumed consent for organ donation, given the overwhelming expressions of public support for transplantation. Ongoing legislative review in the UK provides an ideal chance to alter the default position to one where potential donors can simply acquiesce or opt out of donation. Combined with consultation with their relatives, this could be a much better method of realising individuals’ wishes. It would also achieve a better balance between the duties owed to the deceased and those owed to people awaiting a transplant.
Modified mandated choice for organ procurement
Authors: Chouhan P , Draper H
Published: June 2003

Presumed consent to organ donation looks increasingly unlikely to be a palatable option for increasing organ procurement in the UK following the publication of the report into events at Alder Hey and elsewhere. Yet, given that the alternative to increasing the number of cadaveric organs available is either to accept a greater number of live donations, or accept that people will continue to die for the want of an organ, public policy makers remain obliged to consider other means of increasing the procurement rate.

In this paper, we meet the main objections to mandated choice (namely that it undermines autonomy and that mandated donation is preferable). We have modified the traditional approach to mandated choice to take into account the force of the objection that mandated donation is preferable, by accepting that people can and do make bad decisions about organ donation and proposing that all accompanying public education and information about cadaveric donation should be directed in favour of donation.