bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line
Authors: Schalinske Kevin L. , Blemings Kenneth P. , Steffen Daniel W. , Chen Opal S. , Eisenstein Richard S.
Published: July 2002

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.
A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified
Xenopus
egg extracts
Authors: Desai Arshad , Deacon Heather W. , Walczak Claire E. , Mitchison Timothy J.
Published: July 2002

Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on
in vitro
-condensed chromosomes. The source of the kinetochore components is a clarified cell extract from
Xenopus
eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein–protein interactions involved in kinetochore assembly and function.
G
i
regulation of secretory vesicle swelling examined by atomic force microscopy
Authors: Jena Bhanu P. , Schneider Stefan W. , Geibel John P. , Webster Paul , Oberleithner Hans , Sritharan Kumudesh C.
Published: July 2002

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the α subunit of a heterotrimeric GTP binding protein G
α
i3
and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15–25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate G
i
proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca
2+
had no effect on zymogen granule size. Taken together, the results indicate that G
α
i3
protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.
Plasmon analyses of
Triticum
(wheat) and
Aegilops
: PCR–single-strand conformational polymorphism (PCR-SSCP) analyses of organellar DNAs
Authors: Wang Gui-Zhi , Miyashita Naohiko T. , Tsunewaki Koichiro
Published: July 2002

To investigate phylogenetic relationships among plasmons in
Triticum
and
Aegilops
, PCR–single-strand conformational polymorphism (PCR-SSCP) analyses were made of 14.0-kb chloroplast (ct) and 13.7-kb mitochondrial (mt)DNA regions that were isolated from 46 alloplasmic wheat lines and one euplasmic line. These plasmons represent 31 species of the two genera. The ct and mtDNA regions included 10 and 9 structural genes, respectively. A total of 177 bands were detected, of which 40.6% were variable. The proportion of variable bands in ctDNA (51.1%) was higher than that of mtDNA (28.9%). The phylogenetic trees of plasmons, derived by two different models, indicate a common picture of plasmon divergence in the two genera and suggest three major groups of plasmons (Einkorn,
Triticum
, and
Aegilops
). Because of uniparental plasmon transmission, the maternal parents of all but one polyploid species were identified. Only one
Aegilops
species,
Ae. speltoides
, was included in the
Triticum
group, suggesting that this species is the plasmon and B and G genome donor of all polyploid wheats. ctDNA variations were more intimately correlated with vegetative characters, whereas mtDNA variations were more closely correlated with reproductive characters. Plasmon divergence among the diploids of the two genera largely paralleled genome divergence. The relative times of origin of the polyploid species were inferred from genetic distances from their putative maternal parents.
Functional interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony formation
Authors: Wu Hong , Klingmüller Ursula , Acurio Adriana , Hsiao Jonathan G. , Lodish Harvey F.
Published: July 2002

Production of mature erythrocytes requires multiple growth factors, but we do not know how their actions are coordinated. Here we show that erythroid progenitors from erythropoietin receptor (Epo-R)
−/−
fetal livers, infected
in vitro
with a retrovirus expressing the wild-type Epo-R, require addition of both Epo and stem cell factor (SCF) to form colony-forming unit erythroid (CFU-E) colonies. Thus, a functional interaction between KIT and the Epo-R, similar to what we reported in cultured cells, is essential for the function of CFU-E progenitors. In contrast, CFU-E colony formation
in vitro
by normal fetal liver progenitors requires only Epo; the essential interaction between activated KIT and the Epo-R must have occurred
in vivo
before or at the CFU-E progenitor stage. Using truncated dominant-negative mutant Epo-Rs, we show that KIT does not activate the Epo-R by inducing its dimerization, but presumably does so by phosphorylating tyrosine residue(s) in its cytosolic domain. By expressing mutant Epo-Rs containing only one of eight cytosolic tyrosines, we show that either tyrosine residue Y464 or Y479 suffices for Epo-dependent cell proliferation. However, only Epo-R F7Y479 is capable of supporting erythroid colony formation when expressed in Epo-R
−/−
fetal liver cells, indicating that Y464 either cannot send a differentiation signal or fails to respond to SCF/KIT activation. This work employs a novel experimental system to study the function of growth factors and their receptors in normal hematopoiesis.
Identification of a functional water channel in cytochrome P450 enzymes
Authors: Oprea Tudor I. , Hummer Gerhard , García Angel E.
Published: July 2002

Cytochrome P450 enzymes are monooxygenases that contain a functional heme b group linked to a conserved cysteine with a thiolate bond. In the native state, the central iron atom is hexacoordinated with a covalently bound water molecule. The exclusion of solvent molecules from the active site is essential for efficient enzymatic function. Upon substrate binding, water has to be displaced from the active site to prevent electron uncoupling that results in hydrogen peroxide or water. In contrast to typical hemoproteins, the protein surface is not directly accessible from the heme of cytochromes P450. We postulate a two-state model in which a conserved arginine, stabilizing the heme propionate in all known cytochrome P450 crystal structures, changes from the initial, stable side-chain conformation to another rotamer (metastable). In this new state, a functional water channel (aqueduct) is formed from the active site to a water cluster located on the thiolate side of the heme, close to the protein surface. This water cluster communicates with the surface in the closed state and is partly replaced by the flipping arginine side chain in the open state, allowing water molecules to exit to the surface or to reaccess the active site. This two-state model suggests the presence of an exit pathway for water between the active site and the protein surface.
An elementary mode coupling theory of random heteropolymer dynamics
Authors: Takada Shoji , Portman John J. , Wolynes Peter G.
Published: July 2002

The Langevin dynamics of a random heteropolymer and its dynamic
glass transition are studied using elementary mode coupling theory.
Contrary to recent reports using a similar framework, a discontinuous
ergodic–nonergodic phase transition is predicted for all Rouse modes
at a finite temperature
T

A

. For sufficiently
long chains,
T

A

is almost independent of chain
length and is in good agreement with the value previously estimated by
a static replica theory.
Stability of empty and peptide-loaded class II major histocompatibility complex molecules at neutral and endosomal pH: Comparison to class I proteins
Authors: Reich Ziv , Altman John D. , Boniface J. Jay , Lyons Daniel S. , Kozono Haruo , Ogg Graham , Morgan Chantal , Davis Mark M.
Published: July 2002

The structure and thermal stability of empty and peptide-filled forms of the murine class II major histocompatibility complex (MHC) molecule I-Ekwere studied at neutral and mildly acidic pH. The two forms have distinct circular dichroic spectra, suggesting that a conformational change may accompany peptide binding. Thermal stability profiles indicate that binding of peptide significantly increases the thermal stability of the empty heterodimers at both neutral and mildly acidic pH. Free energies calculated from these data provide a direct measure of this stabilization and show that the empty form of I-Ekis significantly more stable than that of class I MHC proteins. Furthermore, for the two MHC class II proteins that were analyzed (I-Ekand I-Ad), thermal stability was not significantly altered by acidification. In contrast, of four class I MHC molecules studied, three have shown a significant loss in complex stability at low pH. The marked stability exhibited by their empty form, as well as their resistance to low pH, as observed in this study, correlate well with the ability of class II MHC molecules to traverse and bind peptides in acidic endosomal vesicles.
Supercoiling-dependent flexibility of adenosine-tract-containing DNA detected by a topological method
Authors: Tsen Hua , Levene Stephen D.
Published: July 2002

Intrinsically bent DNA sequences have been implicated in the activation of transcription by promoting juxtaposition of DNA sequences near the terminal loop of a superhelical domain. We have developed a novel topological assay for DNA looping based on λ integrative recombination to study the effects of intrinsically bent DNA sequences on the tertiary structure of negatively supercoiled DNA. Remarkably, the localization of adenosine-tract (A-tract) sequences in the terminal loop of a supercoiled plasmid is independent of the extent of intrinsic bending. The results suggest that A-tract-containing sequences have other properties that organize the structure of superhelical domains apart from intrinsic bending and may explain the lack of conservation in the degree of A-tract-dependent bending among DNA sequences located upstream of bacterial promoters.
Cytoplasmic sequestration of an
O
6
-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells
Authors: Chen Frank Y. , Harris Linda C. , Remack Joanna S. , Brent Thomas P.
Published: July 2002

O
6
-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O
6
of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer
+
) and -deficient (Mer
−
) cells, correlated with the endogenous MGMT activity in Mer
+
cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5′-CTGGGTCGC-3′) for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer
+
cells tested but was apparently restricted to the cytoplasm of Mer
−
cells. We conclude that the MEBP–enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer
+
cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer
−
cells.
Purification and characterization of a human RNA adenosine deaminase for glutamate receptor B pre-mRNA editing
Authors: Yang Jing-Hua , Sklar Pamela , Axel Richard , Maniatis Tom
Published: July 2002

The glutamate receptor subunit B (GluR-B) pre-mRNA is edited at two adenosine residues, resulting in amino acid changes that alter the electrophysiologic properties of the glutamate receptor. Previous studies showed that these amino acid changes are due to adenosine to inosine conversions in two codons resulting from adenosine deamination. Here, we describe the purification and characterization of an activity from human HeLa cells that efficiently and accurately edits GluR-B pre-mRNA at both of these sites. The purified activity contains a human homolog of the recently reported rat RED1 (rRED1) protein, a member of the family of double-stranded RNA-dependent deaminase proteins. Recombinant human RED1 (hRED1), but not recombinant dsRAD, another member of the family, efficiently edits both the Q/R and R/G sites of GluR-B RNA. We conclude that the GluR-B editing activity present in HeLa cell extracts and the recombinant hRED1 protein are indistinguishable.
Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man
Authors: Cideciyan Artur V. , Zhao Xinyu , Nielsen Lori , Khani Shahrokh C. , Jacobson Samuel G. , Palczewski Krzysztof
Published: July 2002

Rhodopsin kinase (RK), a specialized G-protein-coupled receptor kinase expressed in retina, is involved in quenching of light-induced signal transduction in photoreceptors. The role of RK in recovery after photoactivation has been explored
in vitro
and
in vivo
experimentally but has not been specifically defined in humans. We investigated the effects on human vision of a mutation in the RK gene causing Oguchi disease, a recessively inherited retinopathy.
In vitro
experiments demonstrated that the mutation, a deletion of exon 5, abolishes the enzymatic activity of RK and is likely a null. Both a homozygote and heterozygote with this RK mutation had recovery phase abnormalities of rod-isolated photoresponses by electroretinography (ERG); photoactivation was normal. Kinetics of rod bleaching adaptation by psychophysics were dramatically slowed in the homozygote but normal final thresholds were attained. Light adaptation was normal at low backgrounds but became abnormal at higher backgrounds. A slight slowing of cone deactivation kinetics in the homozygote was detected by ERG. Cone bleaching adaptation and background adaptation were normal. In this human
in vivo
condition without a functional RK and probable lack of phosphorylation and arrestin binding to activated rhodopsin, reduction of photolyzed chromophore and regeneration processes with 11-
cis
-retinal probably constitute the sole pathway for recovery of rod sensitivity. The role of RK in rods would thus be to accelerate inactivation of activated rhodopsin molecules that in concert with regeneration leads to the normal rate of recovery of sensitivity. Cones may rely mainly on regeneration for the inactivation of photolyzed visual pigment, but RK also contributes to cone recovery.
The human homologue of
Saccharomyces cerevisiae
Gle1p is required for poly(A)
+
RNA export
Authors: Watkins Janis L. , Murphy Robert , Emtage Jennifer L. T. , Wente Susan R.
Published: July 2002

The mechanism of mRNA export is a complex issue central to cellular physiology. We characterized previously yeast Gle1p, a protein with a leucine-rich (LR) nuclear export sequence (NES) that is essential for poly(A)
+
RNA export in
Saccharomyces cerevisiae
. To characterize elements of the vertebrate mRNA export pathway, we identified a human homologue of yeast Gle1p and analyzed its function in mammalian cells.
hGLE1
encodes a predicted 75-kDa polypeptide with high sequence homology to yeast Gle1p, but hGle1p does not contain a sequence motif matching any of the previously characterized NESs.
hGLE1
can complement a yeast
gle1
temperature-sensitive export mutant only if a LR-NES is inserted into it. To determine whether hGle1p played a role in nuclear export, anti-hGle1p antibodies were microinjected into HeLa cells.
In situ
hybridization of injected cells showed that poly(A)
+
RNA export was inhibited. In contrast, there was no effect on the nuclear import of a glucocorticoid receptor reporter. We conclude that hGle1p functions in poly(A)
+
RNA export, and that human cells facilitate such export with a factor similar to yeast but without a recognizable LR-NES. With hGle1p localized at the nuclear pore complexes, hGle1p is positioned to act at a terminal step in the export of mature RNA messages to the cytoplasm.
Low-temperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal
Authors: Andrews Matthew T. , Squire Teresa L. , Bowen Christopher M. , Rollins Martha B.
Published: July 2002

Hibernation is a physiological adaptation characterized by dramatic decreases in heart rate, body temperature, and metabolism, resulting in long-term dormancy. Hibernating mammals survive for periods up to 6 mo in the absence of food by minimizing carbohydrate catabolism and using triglyceride stores as their primary source of fuel. The cellular and molecular mechanisms underlying the changes from a state of activity to the hibernating state are poorly understood; however, the selective expression of genes offers one level of control. To address this problem, we used a differential gene expression screen to identify genes that are responsible for the physiological characteristics of hibernation in the heart of the thirteen-lined ground squirrel (
Spermophilus tridecemlineatus
). Here, we report that genes for pancreatic lipase and pyruvate dehydrogenase kinase isozyme 4 are up-regulated in the heart during hibernation. Pancreatic lipase is normally expressed exclusively in the pancreas, but when expressed in the hibernating heart it liberates fatty acids from triglycerides at temperatures as low as 0°C. Pyruvate dehydrogenase kinase isozyme 4 inhibits carbohydrate oxidation and depresses metabolism by preventing the conversion of pyruvate to Ac-CoA. The resulting anaerobic glycolysis and low-temperature lipid catabolism provide evidence that adaptive changes in cardiac physiology are controlled by the differential expression of genes during hibernation.
Detection of subtle phenotypes: The case of the cell adhesion molecule csA in
Dictyostelium
Authors: Ponte Eleonora , Bracco Enrico , Faix Jan , Bozzaro Salvatore
Published: July 2002

Dictyostelium
amoebae aggregate into a multicellular organism by cAMP-driven chemotaxis and cell–cell adhesion. Cell adhesion is mediated by an EDTA-sensitive and an EDTA-resistant adhesion system. The latter is developmentally regulated and triggered by homophilic interactions of the membrane glycoprotein csA; on disruption of the encoding gene, EDTA-resistant contacts fail to form. Nevertheless, csA-null cells under usual laboratory conditions aggregate normally and complete development. By using experimental conditions that reproduce more closely the habitat of
Dictyostelium
amoebae, evidence is provided that csA is required for development and that its expression confers a selective advantage to populations of wild-type cells over csA-null mutants. The latter display reduced cell–cell adhesion, increased adhesiveness to the substratum, and slower motility, which lead to their sorting out from aggregating wild-type cells. It is proposed that the experimental conditions commonly used in the laboratory are not stringent enough to assess the developmental role of csA and other proteins. The assay described can be used to detect subtle phenotypes, to reexamine the developmental role of apparently nonessential genes, and to test the validity of recent models on emergence and maintenance of apparent genetic redundancy.
Presence of a gene encoding choline sulfatase in
Sinorhizobium meliloti bet
operon: Choline-
O
-sulfate is metabolized into glycine betaine
Authors: Østerås Magne , Boncompagni Eric , Vincent Nadine , Poggi Marie-Christine , Le Rudulier Daniel
Published: July 2002

Glycine betaine is a potent osmoprotectant accumulated by
Sinorhizobium meliloti
to cope with osmotic stress. The biosynthesis of glycine betaine from choline is encoded by an operon of four genes,
betICBA
, as determined by sequence and mutant analysis. The
betI
and
betC
genes are separated by an intergenic region containing a 130-bp mosaic element that also is present between the
betB
and
betA
genes. In addition to the genes encoding a presumed regulatory protein (
betI
), the betaine aldehyde dehydrogenase (
betB
), and the choline dehydrogenase (
betA
) enzymes also found in
Escherichia coli
, a new gene (
betC
) was identified as encoding a choline sulfatase catalyzing the conversion of choline-
O
-sulfate and, at a lower rate, phosphorylcholine, into choline. Choline sulfatase activity was absent from
betC
but not from
betB
mutants and was shown to be induced indifferently by choline or choline-
O
-sulfate as were the other enzymes of the pathway. Unlike what has been shown in other bacteria and plants, choline-
O
-sulfate is not used as an osmoprotectant
per se
in
S. meliloti
, but is metabolized into glycine betaine.
S. meliloti
also can use this compound as the sole carbon, nitrogen, and sulfur source for growth and that depends on a functional
bet
locus. In conclusion, choline-
O
-sulfate and phosphorylcholine, which are found in higher plants and fungi, appear to be substrates for glycine betaine biosynthesis in
S. meliloti
.
Habitat structure determines competition intensity and invasion success in gecko lizards
Authors: Petren Kenneth , Case Ted J.
Published: July 2002

Species diversity is correlated with structural complexity in many animal communities; however, experimental tests of the mechanisms underlying this important relationship are rare, especially in terrestrial communities. We manipulated physical features of the habitat of gecko lizards and measured the effect on exploitation competition for insects. Increasing both the dispersion of food resources and microhabitat topography dramatically reduced interspecific competition. Adding topographic structure reduced the advantages of the larger, faster, invasive species. Interindividual spacing decreased, but intraspecific agonistic interference increased in the more territorial, resident species. Human structural alterations of the environment facilitate invasion and competitive displacement in this system. Physical microhabitat structure can potentially affect species interactions through a variety of complex mechanisms.
Consequences of exclusive expression
in vivo
of kit-ligand lacking the major proteolytic cleavage site
Authors: Tajima Youichi , Moore Malcolm A. S. , Soares Vera , Ono Masao , Kissel Holger , Besmer Peter
Published: July 2002

Membrane growth factors that are processed to produce soluble ligands may function both as soluble factors and as membrane factors. The membrane growth factor Kit-ligand (KL), the ligand of the Kit receptor tyrosine kinase, is encoded at the
Sl
locus, and mice carrying
Sl
mutations have defects in hematopoiesis, gametogenesis, and melanogenesis. Two alternatively spliced KL transcripts encode two cell-associated KL protein products, KL-1 and KL-2. The KL-2 protein lacks the major proteolytic cleavage site for the generation of soluble KL, thus representing a more stable cell-associated form of KL. We investigated the consequences of exclusive expression of KL-2
in vivo
. The KL gene in embryonic stem cells was modified and KL exon 6 was replaced with a PGKneoNTRtkpA cassette by homologous recombination, and mice carrying the

Sl
KL2

allele were obtained.

Sl
KL2
/Sl
KL2

mice had only slightly reduced levels of soluble KL in their serum, suggesting that
in vivo
KL-2 may be processed to produce soluble KL-2S. The steady-state characteristics of the hematopoietic system and progenitor numbers were normal, and the mutant animals were not anemic. However, mast cell numbers in the skin and peritoneum were reduced and the mutant animals displayed increased sensitivity to sublethal doses of γ-irradiation. Therefore, KL-2 may substitute for KL-1 in most situations with the exception of the production of mast cells, and induced proteolytic cleavage of KL-1 to produce soluble KL may have a role in the regeneration of hematopoietic tissue after radiation injury.
Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45
Authors: Zhang Jianhua , Liu Xuesong , Scherer David C. , van Kaer Luc , Wang Xiaodong , Xu Ming
Published: July 2002

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric nuclease complex critical for the induction of DNA fragmentationin vitro. To understand thein vivorole of DFF45 in programmed cell death, we generated DFF45 mutant mice. DNA fragmentation activity is completely abolished in cell extracts from DFF45 mutant tissues. In response to apoptotic stimuli, splenocytes, thymocytes, and granulocytes from DFF45 mutant mice are resistant to DNA fragmentation, and splenocytes and thymocytes are also resistant to chromatin condensation. Nevertheless, development of the immune system in the DFF45 mutant mice is normal. These results demonstrate that DFF45 is critical for the induction of DNA fragmentation and chromatin condensationin vivo, but is not required for normal immune system development.
Characterization of the
Saccharomyces cerevisiae ERG26
gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis
Authors: Gachotte D. , Barbuch R. , Gaylor J. , Nickel E. , Bard M.
Published: July 2002

All but two genes involved in the ergosterol biosynthetic pathway in
Saccharomyces cerevisiae
have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (
ERG25
) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the
Nocardia sp
NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa
Saccharomyces
ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a
hem3
mutation. The YGL001c (
ERG26
) disruption also was viable in a
hem 1
Δ strain grown in the presence of ergosterol. Introduction of the
erg26
mutation into an
erg1
(squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that
erg26
mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4β,14α-dimethyl-4α-carboxy-cholesta-8,24-dien-3β-ol and 4β-methyl-4α-carboxy-cholesta-8,24-dien-3β-ol, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent
erg26
strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.
Models of immune memory: On the role of cross-reactive stimulation, competition, and homeostasis in maintaining immune memory
Authors: Antia Rustom , Pilyugin Sergei S. , Ahmed Rafi
Published: July 2002

There has been much debate on the contribution of processes such as the persistence of antigens, cross-reactive stimulation, homeostasis, competition between different lineages of lymphocytes, and the rate of cell turnover on the duration of immune memory and the maintenance of the immune repertoire. We use simple mathematical models to investigate the contributions of these various processes to the longevity of immune memory (defined as the rate of decline of the population of antigen-specific memory cells). The models we develop incorporate a large repertoire of immune cells, each lineage having distinct antigenic specificities, and describe the dynamics of the individual lineages and total population of cells. Our results suggest that, if homeostatic control regulates the total population of memory cells, then, for a wide range of parameters, immune memory will be long-lived in the absence of persistent antigen (
T
1/2
> 1 year). We also show that the longevity of memory in this situation will be insensitive to the relative rates of cross-reactive stimulation, the rate of turnover of immune cells, and the functional form of the term for the maintenance of homeostasis.
Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in
Escherichia coli
Authors: Mukherjee Amit , Cao Chune , Lutkenhaus Joe
Published: July 2002

The bacterial cell division protein FtsZ assembles into the cytokinetic Z ring that directs cytokinesis in prokaryotes. In
Escherichia coli
the formation of the Z ring is prevented by induction of the cell division inhibitor SulA (SfiA), a component of the SOS response. Here we show that a MalE-SulA fusion that retains this inhibitory function
in vivo
inhibits the GTPase activity and polymerization of FtsZ
in vitro
. MalE-SulA10, which does not block Z ring formation
in vivo
, is unable to inhibit the GTPase activity and polymerization
in vitro
. Furthermore, FtsZ114, which is refractory to SulA
in vivo
, is not inhibited by MalE-SulA. These results indicate that SulA blocks Z ring formation by blocking FtsZ polymerization.
A role for histone deacetylase activity in HDAC1-mediated transcriptional repression
Authors: Hassig Christian A. , Tong Jeffrey K. , Fleischer Tracey C. , Owa Takashi , Grable Phyllis G. , Ayer Donald E. , Schreiber Stuart L.
Published: July 2002

Treatment of mammalian cells with small molecule histone deacetylase (HDAC) inhibitors induces changes in the transcription of specific genes. These changes correlate directly with an increase in the acetylation levels of all four core histones
in vivo
. Antibodies directed against endogenous HDAC1, HDAC2, or HDAC3 immunoprecipitate histone deacetylase activity that is inhibited
in vitro
by the small molecule trapoxin (TPX), and all three HDACs are retained by a TPX-affinity matrix. HDAC1 and HDAC2 are associated in HeLa cells in a complex that is predominantly separate from an HDAC3 immune complex. Both Jurkat HDAC1 and HeLa HDAC1/2 immune complexes deacetylate all four core histones and recombinant HDAC1 deacetylates free and nucleosomal histones
in vitro
. Purified recombinant HDAC1 deacetylates core histones in the absence of protein cofactors. Site-directed mutagenesis was used to identify residues required for the enzymatic and structural integrity of HDAC1. Mutation of any one of four conserved residues causes deleterious effects on deacetylase activity and a reduced ability to bind a TPX-affinity matrix. A subset of these mutations also cause a decreased interaction with the HDAC1-associated proteins RbAp48 and mSin3A. Disruption of histone deacetylase activity either by TPX or by direct mutation of a histidine presumed to be in the active site abrogates HDAC1-mediated transcriptional repression of a targeted reporter gene
in vivo
.
The underlying pathway structure of biochemical reaction networks
Authors: Schilling Christophe H. , Palsson Bernhard O.
Published: July 2002

Bioinformatics is yielding extensive, and in some cases complete, genetic and biochemical information about individual cell types and cellular processes, providing the composition of living cells and the molecular structure of its components. These components together perform integrated cellular functions that now need to be analyzed. In particular, the functional definition of biochemical pathways and their role in the context of the whole cell is lacking. In this study, we show how the mass balance constraints that govern the function of biochemical reaction networks lead to the translation of this problem into the realm of linear algebra. The functional capabilities of biochemical reaction networks, and thus the choices that cells can make, are reflected in the null space of their stoichiometric matrix. The null space is spanned by a finite number of basis vectors. We present an algorithm for the synthesis of a set of basis vectors for spanning the null space of the stoichiometric matrix, in which these basis vectors represent the underlying biochemical pathways that are fundamental to the corresponding biochemical reaction network. In other words, all possible flux distributions achievable by a defined set of biochemical reactions are represented by a linear combination of these basis pathways. These basis pathways thus represent the underlying pathway structure of the defined biochemical reaction network. This development is significant from a fundamental and conceptual standpoint because it yields a holistic definition of biochemical pathways in contrast to definitions that have arisen from the historical development of our knowledge about biochemical processes. Additionally, this new conceptual framework will be important in defining, characterizing, and studying biochemical pathways from the rapidly growing information on cellular function.
Regulation of thalamic neurite outgrowth by the Eph ligand ephrin-A5: Implications in the development of thalamocortical projections
Authors: Gao Pan-Pan , Yue Yong , Zhang Jian-Hua , Cerretti Douglas Pat , Levitt Pat , Zhou Renping
Published: July 2002

The cerebral cortex is parcellated into different functional domains that receive distinct inputs from other cortical and subcortical regions. The molecular mechanisms underlying the specificity of connections of cortical afferents remain unclear. We report here that the Eph family tyrosine kinase receptor EphA5 and the ligand ephrin-A5 may play a key role in the exclusion of the limbic thalamic afferents from the sensorimotor cortex by mediating repulsive interactions.
In situ
hybridization shows that the EphA5 transcript is expressed at high levels in both cortical and subcortical limbic regions, including the frontal cortex, the subiculum, and the medial thalamic nuclei. In contrast, ephrin-A5 is transcribed abundantly in the sensorimotor cortex. Consistent with the complementary expression, the ligand inhibited dramatically the growth of neurites from neurons isolated from the medial thalamus but was permissive for the growth of neurites from lateral thalamic neurons, which is primarily nonlimbic. Similarly, the growth of neurites from Eph-A5-expressing neurons isolated from the subiculum was inhibited by ephrin-A5. Our studies suggest that the Eph family ligand ephrin-A5 serves as a general inhibitor of axonal growth from limbic neurons, which may serve to prevent innervation of inappropriate primary sensorimotor regions, thus contributing to the generation of specificity of thalamic cortical afferents.
Magnesium-dependent folding of self-splicing RNA: Exploring the link between cooperativity, thermodynamics, and kinetics
Authors: Pan Jie , Thirumalai D. , Woodson Sarah A.
Published: July 2002

Folding of the
Tetrahymena
self-splicing RNA into its active conformation involves a set of discrete intermediate states. The Mg
2+
-dependent equilibrium transition from the intermediates to the native structure is more cooperative than the formation of the intermediates from the unfolded states. We show that the degree of cooperativity is linked to the free energy of each transition and that the rate of the slow transition from the intermediates to the native state decreases exponentially with increasing Mg
2+
concentration. Monovalent salts, which stabilize the folded RNA nonspecifically, induce states that fold in less than 30 s after Mg
2+
is added to the RNA. A simple model is proposed that predicts the folding kinetics from the Mg
2+
-dependent change in the relative stabilities of the intermediate and native states.
Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene
Authors: Rocklin Amy M. , Tierney David L. , Kofman Victoria , Brunhuber Norbert M. W. , Hoffman Brian M. , Christoffersen Rolf E. , Reich Norbert O. , Lipscomb John D. , Que Lawrence
Published: July 2002

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O
2
, CO
2
, and ascorbate are required for
in vitro
enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme⋅(substrate or cofactor)⋅NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O
2
complexes. It is shown here that the ternary Fe(II)ACCO⋅ACC⋅NO complex is readily formed, but no Fe(II)ACCO⋅ascorbate⋅NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with
15
N or
17
O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the α-amino and α-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O
2
bind to the iron as a step in O
2
activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O
2
, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.
A γ-herpesvirus sneaks through a CD8+T cell response primed to a lytic-phase epitope
Authors: Stevenson Philip G. , Belz Gabrielle T. , Castrucci Maria R. , Altman John D. , Doherty Peter C.
Published: July 2002

To determine whether established CD8+T cell memory to an epitope prominent during the replicative phase of a γ-herpesvirus infection protects against subsequent challenge, mice were primed with a recombinant vaccinia virus expressing the p56 peptide and then boosted by intranasal exposure to an influenza A virus incorporating p56 in the neuraminidase protein. Clonally expanded populations of functional, p56-specific CD8+T cells were present at high frequency in both the lung and the lymphoid tissue 1 month later, immediately before respiratory challenge with γHV-68. This prime-and-boost regime led to a massive reduction of productive γHV-68 infection in the respiratory tract and, initially, to much lower levels of latency in both the regional lymph nodes and the spleen. The CD8+T cell response to another epitope (p79) was diminished, there was less evidence of B cell activation, and the onset of the CD4+T cell-dependent splenomegaly was delayed. Within 3–4 weeks of the γHV-68 challenge, however, the extent of latent infection in the lymph nodes and spleen was equivalent, and both groups developed the prominent infectious mononucleosis-like syndrome that is characteristic of this infection. The reverse protocol (influenza then vaccinia) seemed to be slightly less effective. Even though immune CD8+T cells may be present at the time and site of virus challenge, establishing a high level of CD8+T cell memory to lytic-phase epitopes alone does not protect against the longer-term consequences of this γHV infection.
The
Arabidopsis SKP1-LIKE1
gene is essential for male meiosis and may control homologue separation
Authors: Yang Ming , Hu Yi , Lodhi Muhammad , McCombie W. Richard , Ma Hong
Published: July 2002

The yeast and human
SKP1
genes regulate the mitotic cell cycle but are not yet known to be required for meiosis. Nine
Arabidopsis SKP1
homologues have been uncovered and are named
ASK1
through
ASK9
. Here, we report the isolation and characterization of a male sterile
Arabidopsis
mutant and show that the mutant defect was caused by a
Ds
transposon insertion into the
ASK1
gene. In the
ask1-1
mutant, abnormal microspores exhibit a range of sizes. Furthermore, during mutant male meiosis, although homologous chromosome pairing appeared normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended. Therefore, in
ask1-1
, at least some homologues remain associated after metaphase I. In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect. Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, we propose that
ASK1
controls homologue separation by degrading or otherwise removing a protein that is required directly or indirectly for homologue association before anaphase I.
Continuous and lurching traveling pulses in neuronal networks with delay and spatially decaying connectivity
Authors: Golomb David , Ermentrout G. Bard
Published: July 2002

Propagation of discharges in cortical and thalamic systems, which is used as a probe for examining network circuitry, is studied by constructing a one-dimensional model of integrate-and-fire neurons that are coupled by excitatory synapses with delay. Each neuron fires only one spike. The velocity and stability of propagating continuous pulses are calculated analytically. Above a certain critical value of the constant delay, these pulses lose stability. Instead, lurching pulses propagate with discontinuous and periodic spatio-temporal characteristics. The parameter regime for which lurching occurs is strongly affected by the footprint (connectivity) shape; bistability may occur with a square footprint shape but not with an exponential footprint shape. For strong synaptic coupling, the velocity of both continuous and lurching pulses increases logarithmically with the synaptic coupling strength
g
syn
for an exponential footprint shape, and it is bounded for a step footprint shape. We conclude that the differences in velocity and shape between the front of thalamic spindle waves
in vitro
and cortical paroxysmal discharges stem from their different effective delay; in thalamic networks, large effective delay between inhibitory neurons arises from their effective interaction via the excitatory cells which display postinhibitory rebound.
A complex ligase ribozyme evolved
in vitro
from a group I ribozyme domain
Authors: Jaeger Luc , Wright Martin C. , Joyce Gerald F.
Published: July 2002

Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an
in vitro
evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10
16
different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3′,5′-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3′ hydroxyl and the other a 5′ triphosphate. Ligation occurs in the context of a Watson–Crick duplex, with a catalytic rate of 0.26 min
−1
under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.
ARG1
(Altered Response to Gravity) encodes a DnaJ-like protein that potentially interacts with the cytoskeleton
Authors: Sedbrook John C. , Chen Rujin , Masson Patrick H.
Published: July 2002

Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vector, promoting upward growth for shoots and downward growth for roots. Little is known about the mechanisms underlying gravitropic signal transduction. We found that mutations in the
ARG1
locus of
Arabidopsis thaliana
alter root and hypocotyl gravitropism without affecting phototropism, root growth responses to phytohormones or inhibitors of auxin transport, or starch accumulation. The positional cloning of
ARG1
revealed a DnaJ-like protein containing a coiled-coil region homologous to coiled coils found in cytoskeleton-interacting proteins. These data suggest that
ARG1
participates in a gravity-signaling process involving the cytoskeleton. A combination of Northern blot studies and analysis of
ARG1-GUS
fusion-reporter expression in transgenic plants demonstrated that
ARG1
is expressed in all organs. Ubiquitous
ARG1
expression in
Arabidopsis
and the identification of an ortholog in
Caenorhabditis elegans
suggest that
ARG1
is involved in other essential processes.