bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid assembly dynamics of the
Escherichia coli
FtsZ-ring demonstrated by fluorescence recovery after photobleaching
Authors: Stricker Jesse , Maddox Paul , Salmon E. D. , Erickson Harold P.
Published: July 2002

FtsZ, the major cytoskeletal component of the bacterial cell-division machine, assembles into a ring (the Z-ring) that contracts at septation. FtsZ is a bacterial homolog of tubulin, with similar tertiary structure, GTP hydrolysis, and
in vitro
assembly. We used green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching to show that the
E. coli
Z-ring is extremely dynamic, continually remodeling itself with a half-time of 30 s. ZipA, a membrane protein involved in cell division that colocalizes with FtsZ, was equally dynamic. The Z-ring of the mutant
ftsZ84
, which has 1/10 the guanosine triphosphatase activity of wild-type FtsZ
in vitro
, showed a 9-fold slower turnover
in vivo
. This finding implies that assembly dynamics are determined primarily by GTP hydrolysis. Despite the greatly reduced assembly dynamics, the
ftsZ84
cells divide with a normal cell-cycle time.
Interferon regulatory factor-3 is an
in vivo
target of DNA-PK
Authors: Karpova Alla Y. , Trost Maren , Murray John M. , Cantley Lewis C. , Howley Peter M.
Published: July 2002

Eukaryotic cells have evolved complex signaling networks to sense environmental stress and to repair stress-induced damage. IFN regulatory factor-3 (IRF-3) is a transcription factor that plays a central role in the host response to viral infection. Although the main activity of IRF-3 characterized to date has been its role in the induction of IFN-α and -β after virus infection, recent evidence indicates additional roles for IRF-3 in the response to DNA damage and in virus-induced apoptosis. Here we identify IRF-3 as the first
in vivo
target for DNA-dependent protein kinase (DNA-PK). Phosphorylation of IRF-3 by DNA-PK after virus infection results in its nuclear retention and delayed proteolysis. These results expand the known roles of DNA-PK and provide a functional link between the cellular machineries that regulate the innate immune response and that sense and respond to DNA damage. As such this study contributes to a more integrated view of the cellular responses to various cellular stress signals.
Homeostatic T cell proliferation in a T cell-dendritic cell coculture system
Authors: Ge Qing , Palliser Deborah , Eisen Herman N. , Chen Jianzhu
Published: July 2002

Naïve T cells do not proliferate in normal individuals in the absence of antigen stimulation, but they proliferate spontaneously when T cells are severely depleted. We show here that coculture of syngeneic dendritic cells (DC) with naïve T cells expressing a single T cell receptor also results in T cell proliferation in the absence of foreign antigen. As in lymphopenic mice, where T cell proliferation depends upon DC, this response in the coculture system requires interaction of the T cells' T cell receptor with self-peptide–MHCs on DC. Thisin vitroproliferation also requires soluble factors, including IL-15 secreted by DC, and can be inhibited potently by cell–cell contact with CD4+CD25+regulatory T cells. The coculture system described may illuminate mechanisms that maintain stable numbers of T cells in normal individuals.
Physical association of ubiquitin ligases and the 26S proteasome
Authors: Xie Youming , Varshavsky Alexander
Published: July 2002

The ubiquitin (Ub) system recognizes degradation signals of the
target proteins through the E3 components of E3-E2 Ub ligases. A
targeted substrate bears a covalently linked multi-Ub chain and is
degraded by the ATP-dependent 26S proteasome, which consists of the 20S
core protease and two 19S particles. The latter mediate the binding and
unfolding of a substrate protein before its transfer to the interior of
the 20S core. It is unclear how a targeted substrate is delivered to
the 26S proteasome, inasmuch as Rpn10p, the only known proteasomal
subunit that binds multi-Ub chains, has been found to be not essential
for degradation of many proteins in the yeast
Saccharomyces
cerevisiae
. Here we show that Ubr1p and Ufd4p, the E3
components of two distinct Ub ligases, directly interact with the 26S
proteasome. Specifically, Ubr1p is shown to bind to the Rpn2p, Rpt1p,
and Rpt6p proteins of the 19S particle, and Ufd4p is shown to bind to
Rpt6p. These and related results suggest that a substrate-bound Ub
ligase participates in the delivery of substrates to the proteasome,
because of affinity between the ligase's E3 component and specific
proteins of the 19S particle.
The
in vivo
function of the ribosome-associated Hsp70, Ssz1, does not require its putative peptide-binding domain
Authors: Hundley Heather , Eisenman Helene , Walter William , Evans Tara , Hotokezaka Yuka , Wiedmann Martin , Craig Elizabeth
Published: July 2002

Two proteins of the Hsp70 class (Ssb and Ssz1) and one of the J-type class (Zuo1) of molecular chaperones reside on the yeast ribosome, with Ssz1 forming a stable heterodimer with Zuo1. We designed experiments to address the roles of these two distantly related ribosome-associated Hsp70s and their functional relationship to Zuo1. Strains lacking all three proteins have the same phenotype as those lacking only one, suggesting that these chaperones all function in the same pathway. The Hsp70 Ssb, whose peptide-binding domain is essential for its
in vivo
function, can be crosslinked to nascent chains on ribosomes that are as short as 54 amino acids, suggesting that Ssb interacts with nascent chains that extend only a short distance beyond the tunnel of the ribosome. A
ssz1
mutant protein lacking its putative peptide-binding domain allows normal growth. Thus, binding of unfolded protein substrates in a manner similar to that of typical Hsp70s is not critical for Ssz1's
in vivo
function. The three chaperones are present in cells in approximately equimolar amounts compared with ribosomes. The level of Ssb can be reduced only a few-fold before growth is affected. However, a 50- to 100-fold reduction of Ssz1 and Zuo1 levels does not have a substantial effect on cell growth. On the basis of these results, we propose that Ssbs function as the major Hsp70 chaperone for nascent chains on the ribosome, and that Ssz1 has evolved to perform a nonclassical function, perhaps modulating Zuo1's ability to function as a J-type chaperone partner of Ssb.
Panhandle and reverse-panhandle PCR enable cloning of der(11) and der(other) genomic breakpoint junctions ofMLLtranslocations and identify complex translocation ofMLL,AF-4, andCDK6
Authors: Raffini Leslie J. , Slater Diana J. , Rappaport Eric F. , Lo Nigro Luca , Cheung Nai-Kong V. , Biegel Jaclyn A. , Nowell Peter C. , Lange Beverly J. , Felix Carolyn A.
Published: July 2002

We used panhandle PCR to clone the der(11) genomic breakpoint junction in three leukemias with t(4;11) and devised reverse-panhandle PCR to clone the breakpoint junction of the other derivative chromosome. This work contributes two elements to knowledge onMLLtranslocations. First is reverse-panhandle PCR for cloning breakpoint junctions of the other derivative chromosomes, sequences of which are germane to understanding theMLLtranslocation process. The technique revealed duplicated sequences in one case of infant acute lymphoblastic leukemia (ALL) and small deletions in a case of treatment-related ALL. The second element is discovery of a three-way rearrangement ofMLL,AF-4, andCDK6in another case of infant ALL. Cytogenetic analysis was unsuccessful at diagnosis, but suggested t(4;11) and del(7)(q21q31) at relapse. Panhandle PCR analysis of the diagnostic marrow identified a breakpoint junction ofMLLintron 8 andAF-4intron 3. Reverse-panhandle PCR identified a breakpoint junction ofCDK6from band 7q21-q22 andMLLintron 9.CDK6encodes a critical cell cycle regulator and is the first gene of this type disrupted byMLLtranslocation. Cdk6 is overexpressed or disrupted by translocation in many cancers. The in-frameCDK6-MLLtranscript is provocative with respect to a potential contribution of the predicted Cdk6-MLL fusion protein in the genesis of the ALL, which also contains an in-frameMLL-AF4transcript. The sequences in these three cases show additionalMLLgenomic breakpoint heterogeneity. Each breakpoint junction suggests nonhomologous end joining and is consistent with DNA damage and repair.CDK6-MLLis a new fusion of both genes.
Genome organization in dicots: Genome duplication in
Arabidopsis
and synteny between soybean and
Arabidopsis
Authors: Grant David , Cregan Perry , Shoemaker Randy C.
Published: July 2002

Synteny between soybean and
Arabidopsis
was studied by using conceptual translations of DNA sequences from loci that map to soybean linkage groups A2, J, and L. Synteny was found between these linkage groups and all four of the
Arabidopsis
chromosomes, where GenBank contained enough sequence for synteny to be identified confidently. Soybean linkage group A2 (soyA2) and
Arabidopsis
chromosome I showed significant synteny over almost their entire lengths, with only 2–3 chromosomal rearrangements required to bring the maps into substantial agreement. Smaller blocks of synteny were identified between soyA2 and
Arabidopsis
chromosomes IV and V (near the
RPP
5 and
RPP
8 genes) and between soyA2 and
Arabidopsis
chromosomes I and V (near the
Phy
A and
Phy
C genes). These subchromosomal syntenic regions were themselves homeologous, suggesting that
Arabidopsis
has undergone a number of segmental duplications or possibly a complete genome duplication during its evolution. Homologies between the homeologous soybean linkage groups J and L and
Arabidopsis
chromosomes II and IV also revealed evidence of segmental duplication in
Arabidopsis
. Further support for this hypothesis was provided by the observation of very close linkage in
Arabidopsis
of homologs of soybean
Vsp
27 and Bng181 (three locations) and purple acid phosphatase-like sequences and homologs of soybean A256 (five locations). Simulations show that the synteny and duplications we report are unlikely to have arisen by chance during our analysis of the homology reports.
Identification of a sequence element directing a protein to nuclear speckles
Authors: Eilbracht Jens , Schmidt-Zachmann Marion S.
Published: July 2002

SF3b
155
is an essential spliceosomal protein, highly
conserved during evolution. It has been identified as a subunit of
splicing factor SF3b, which, together with a second multimeric complex
termed SF3a, interacts specifically with the 12S U2 snRNP and converts
it into the active 17S form. The protein displays a characteristic
intranuclear localization. It is diffusely distributed in the
nucleoplasm but highly concentrated in defined intranuclear structures
termed “speckles,” a subnuclear compartment enriched in small
ribonucleoprotein particles and various splicing factors. The primary
sequence of SF3b
155
suggests a multidomain structure,
different from those of other nuclear speckles components. To identify
which part of SF3b
155
determines its specific intranuclear
localization, we have constructed expression vectors encoding a series
of epitope-tagged SF3b
155
deletion mutants as well as
chimeric combinations of SF3b
155
sequences with the soluble
cytoplasmic protein pyruvate kinase. Following transfection of cultured
mammalian cells, we have identified (i) a functional nuclear
localization signal of the monopartite type (KRKRR, amino acids
196–200) and (ii) a molecular segment with multiple threonine-proline
repeats (amino acids 208–513), which is essential and sufficient to
confer a specific accumulation in nuclear speckles. This latter
sequence element, in particular amino acids 208–440, is required for
correct subcellular localization of SF3b
155
and is also
sufficient to target a reporter protein to nuclear speckles. Moreover,
this “speckle-targeting sequence” transfers the capacity for
interaction with other U2 snRNP components.
Human receptors for sweet and umami taste
Authors: Li Xiaodong , Staszewski Lena , Xu Hong , Durick Kyle , Zoller Mark , Adler Elliot
Published: July 2002

The three members of the T1R class of taste-specific G protein-coupled receptors have been hypothesized to function in combination as heterodimeric sweet taste receptors. Here we show that human T1R2/T1R3 recognizes diverse natural and synthetic sweeteners. In contrast, human T1R1/T1R3 responds to the umami taste stimulus
l
-glutamate, and this response is enhanced by 5′-ribonucleotides, a hallmark of umami taste. The ligand specificities of rat T1R2/T1R3 and T1R1/T1R3 correspond to those of their human counterparts. These findings implicate the T1Rs in umami taste and suggest that sweet and umami taste receptors share a common subunit.
Toc34 is a preprotein receptor regulated by GTP and phosphorylation
Authors: Sveshnikova Natalia , Soll Jürgen , Schleiff Enrico
Published: July 2002

Most proteins present in chloroplasts are synthesized in the cytosol and are posttranslationally translocated into the organelle. A multicomponent translocation machinery located in both the outer and the inner envelope of chloroplasts was identified, but the mode of action of many subunits remains unclear. Here, we describe the regulation of an early step of translocation occurring at the outer envelope. The outer envelope translocon subunit Toc34 can be phosphorylated, and GTP binding is regulated by phosphorylation.
In vitro
, Toc34 acts as a receptor for proteins containing a chloroplast-targeting signal. Interaction of Toc34 with the transit peptide is highly regulated and depends on GTP binding to Toc34 and on phosphorylation of the transit peptide of the preprotein.
Complex mtDNA constitutes an approximate 620-kb insertion on
Arabidopsis thaliana
chromosome 2: Implication of potential sequencing errors caused by large-unit repeats
Authors: Stupar Robert M. , Lilly Jason W. , Town Christopher D. , Cheng Zhukuan , Kaul Samir , Buell C. Robin , Jiang Jiming
Published: July 2002

Previously conducted sequence analysis of
Arabidopsis
thaliana
(ecotype Columbia-0) reported an insertion of 270-kb
mtDNA into the pericentric region on the short arm of chromosome 2. DNA
fiber-based fluorescence
in situ
hybridization analyses
reveal that the mtDNA insert is 618 ± 42 kb, ≈2.3 times greater
than that determined by contig assembly and sequencing analysis.
Portions of the mitochondrial genome previously believed to be absent
were identified within the insert. Sections of the mtDNA are repeated
throughout the insert. The cytological data illustrate that DNA contig
assembly by using bacterial artificial chromosomes tends to produce a
minimal clone path by skipping over duplicated regions, thereby
resulting in sequencing errors. We demonstrate that fiber-fluorescence
in situ
hybridization is a powerful technique to analyze
large repetitive regions in the higher eukaryotic genomes and is a
valuable complement to ongoing large genome sequencing projects.
Ser
67
-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor
Authors: Huang Ke-xue , Paudel Hemant K.
Published: July 2002

Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated
in vivo
on Thr
35
and Ser
67
. Thr
35
is phosphorylated by cAMP-dependent protein kinase (A kinase), and Thr
35
-phosphorylated I-1 inhibits PP1. Until now the kinase that phosphorylates Ser
67
had not been identified and the physiological role of Ser
67
phosphorylation was unknown. In this study we detected a high level of kinase activity in brain extract when a glutathione
S
-transferase (GST) fusion I-1 mutant containing an Ala substituted for Thr
35
[GST-I-1(T35A)] was used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein kinase (NCLK) in the brain extract could not be separated from each other by a series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated with anti-NCLK antibody from kinase-active column fractions. Purified NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed mutagenesis determined that NCLK phosphorylates Ser
67
of I-1. NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC
50
values ≈9.5 and 13.8 nM, respectively. When compared, A kinase-phosphorylated I-1 was only ≈1.2 times more inhibitory than NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential
in vivo
I-1 kinase and that Thr
35
and Ser
67
phosphorylation independently activate I-1.
LXRα functions as a cAMP-responsive transcriptional regulator of gene expression
Authors: Tamura Kouichi , Chen Yuqing E. , Horiuchi Masatsugu , Chen Qin , Daviet Laurent , Yang Zhiyong , Daviet Laurent , Lopez-Ilasaca Marco , Mu Hui , Pratt Richard E. , Dzau Victor J.
Published: July 2002

LXRα is a member of a nuclear receptor superfamily that regulates transcription. LXRα forms a heterodimer with RXRα, another member of this family, to regulate the expression of cholesterol 7α-hydroxylase by means of binding to the DR4-type cis-element. Here, we describe a function for LXRα as a cAMP-responsive regulator of renin and c-myc gene transcriptions by the interaction with a specific cis-acting DNA element, CNRE (an overlapping cAMP response element and a negative response element). Our previous studies showed that renin gene expression is regulated by cAMP, at least partly, through the CNRE sequence in its 5′-flanking region. This sequence is also found in c-myc and several other genes. Based on our cloning results using the yeast one-hybrid system, we discovered that the mouse homologue of human LXRα binds to the CNRE and demonstrated that it binds as a monomer. To define the function of LXRα on gene expression, we transfected the renin-producing renal As4.1 cells with LXRα expression plasmid. Overexpression of LXRα in As4.1 cells confers cAMP inducibility to reporter constructs containing the renin CNRE. After stable transfection of LXRα, As4.1 cells show a cAMP-inducible up-regulation of renin mRNA expression. In parallel experiments, we demonstrated that LXRα can also bind to the homologous CNRE in the c-myc promoter. cAMP promotes transcription through c-myc/CNRE:LXRα interaction in LXRα transiently transfected cells and increases c-myc mRNA expression in stably transfected cells. Identification of LXRα as a cAMP-responsive nuclear modulator of renin and c-myc expression not only has cardiovascular significance but may have generalized implication in the regulation of gene transcription.
Frequent fusion of the
JAZF1
and
JJAZ1
genes in endometrial stromal tumors
Authors: Koontz Jason I. , Soreng A. Lee , Nucci Marisa , Kuo Frank C. , Pauwels Patrick , van den Berghe Herman , Cin Paola Dal , Fletcher Jonathan A. , Sklar Jeffrey
Published: July 2002

Endometrial stromal tumors are divided into three types: benign
stromal nodules, endometrial stromal sarcomas, and undifferentiated
endometrial sarcomas. A variety of cytogenetic abnormalities involving
chromosome 7 have been reported in endometrial stromal sarcomas,
including a recurrent t(7;17)(p15;q21). We have identified two zinc
finger genes, which we have termed
JAZF1
and
JJAZ1
, at the sites of the 7p15 and 17q21 breakpoints.
Analyses of tumor RNA indicate that a
JAZF1
/
JJAZ1
fusion is present in all
types of endometrial stromal tumors; however, the fusion appears to be
rarer among endometrial stromal sarcomas that would be considered
high-grade according to certain classification schemes. These findings
suggest that the less malignant endometrial stromal tumors may evolve
toward more malignant types, but that some endometrial stromal sarcomas
with relatively abundant mitotic activity may compose a biologically
distinct group.
Control of protein crystal nucleation around the metastable liquid–liquid phase boundary
Authors: Galkin Oleg , Vekilov Peter G.
Published: July 2002

The capability to enhance or suppress the nucleation of protein crystals opens opportunities in various fundamental and applied areas, including protein crystallography, production of protein crystalline pharmaceuticals, protein separation, and treatment of protein condensation diseases. Herein, we show that the rate of homogeneous nucleation of lysozyme crystals passes through a maximum in the vicinity of the liquid–liquid phase boundary hidden below the liquidus (solubility) line in the phase diagram of the protein solution. We found that glycerol and polyethylene glycol (which do not specifically bind to proteins) shift this phase boundary and significantly suppress or enhance the crystal nucleation rates, although no simple correlation exists between the action of polyethylene glycol on the phase diagram and the nucleation kinetics. The control mechanism does not require changes in the protein concentration, acidity, and ionicity of the solution. The effects of the two additives on the phase diagram strongly depend on their concentration, which provides opportunities for further tuning of nucleation rates.
Nonapoptotic neurodegeneration in a transgenic mouse model of Huntington's disease
Authors: Turmaine Mark , Raza Aysha , Mahal Amarbirpal , Mangiarini Laura , Bates Gillian P. , Davies Stephen W.
Published: July 2002

Huntington's disease (HD) is a fatal inherited neurodegenerative disorder characterized by personality changes, motor impairment, and subcortical dementia. HD is one of a number of diseases caused by expression of an expanded polyglutamine repeat. We have developed several lines of mice that are transgenic for exon 1 of the HD gene containing an expanded CAG sequence. These mice exhibit a defined neurological phenotype along with neuronal changes that are pathognomonic for the disease. We have previously observed the appearance of neuronal intranuclear inclusions, but did not find evidence for neurodegeneration. In this study, we report that all lines of these mice develop a late onset neurodegeneration within the anterior cingulate cortex, dorsal striatum, and of the Purkinje neurons of the cerebellum. Dying neurons characteristically exhibit neuronal intranuclear inclusions, condensation of both the cytoplasm and nucleus, and ruffling of the plasma membrane while maintaining ultrastructural preservation of cellular organelles. These cells do not develop blebbing of the nucleus or cytoplasm, apoptotic bodies, or fragmentation of DNA. Neuronal death occurs over a period of weeks not hours. We also find degenerating cells of similar appearance within these same regions in brains of patients who had died with HD. We therefore suggest that the mechanism of neuronal cell death in both HD and a transgenic mouse model of HD is neither by apoptosis nor by necrosis.
All kinesin superfamily protein, KIF, genes in mouse and human
Authors: Miki Harukata , Setou Mitsutoshi , Kaneshiro Kiyofumi , Hirokawa Nobutaka
Published: July 2002

Intracellular transport is essential for morphogenesis and functioning of the cell. The kinesin superfamily proteins (KIFs) have been shown to transport membranous organelles and protein complexes in a microtubule- and ATP-dependent manner. More than 30 KIFs have been reported in mice. However, the nomenclature of KIFs has not been clearly established, resulting in various designations and redundant names for a single KIF. Here, we report the identification and classification of all KIFs in mouse and human genome transcripts. Previously unidentified murine KIFs were found by a PCR-based search. The identification of all KIFs was confirmed by a database search of the total human genome. As a result, there are a total of 45 KIFs. The nomenclature of all KIFs is presented. To understand the function of KIFs in intracellular transport in a single tissue, we focused on the brain. The expression of 38 KIFs was detected in brain tissue by Northern blotting or PCR using cDNA. The brain, mainly composed of highly differentiated and polarized cells such as neurons and glia, requires a highly complex intracellular transport system as indicated by the increased number of KIFs for their sophisticated functions. It is becoming increasingly clear that the cell uses a number of KIFs and tightly controls the direction, destination, and velocity of transportation of various important functional molecules, including mRNA. This report will set the foundation of KIF and intracellular transport research.
One-step inactivation of chromosomal genes in
Escherichia coli
K-12 using PCR products
Authors: Datsenko Kirill A. , Wanner Barry L.
Published: July 2002

We have developed a simple and highly efficient method to disrupt chromosomal genes in
Escherichia coli
in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the
arcB
,
cyaA
,
lacZYA
,
ompR
-
envZ
,
phnR
,
pstB
,
pstCA
,
pstS
,
pstSCAB
-
phoU
,
recA
, and
torSTRCAD
genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of
E. coli
and other bacteria because the procedure can be done in wild-type cells.
A protein required for prion generation: [URE3] induction requires the Ras-regulated Mks1 protein
Authors: Edskes Herman K. , Wickner Reed B.
Published: July 2002

Infectious proteins (prions) can arise
de novo
as well as by transmission from another individual.
De novo
prion generation is believed responsible for most cases of Creutzfeldt–Jakob disease and for initiating the mad cow disease epidemic. However, the cellular components needed for prion generation have not been identified in any system. The [URE3] prion of
Saccharomyces cerevisiae
is an infectious form of Ure2p, apparently a self-propagating amyloid. We now demonstrate a protein required for
de novo
prion generation. Mks1p negatively regulates Ure2p and is itself negatively regulated by the presence of ammonia and by the Ras–cAMP pathway. We find that in
mks1
Δ strains,
de novo
generation of the [URE3] prion is blocked, although [URE3] introduced from another strain is expressed and propagates stably. Ras2
Val19
increases cAMP production and also blocks [URE3] generation. These results emphasize the distinction between prion generation and propagation, and they show that cellular regulatory mechanisms can critically affect prion generation.
The Friend of GATA proteins U-shaped, FOG-1, and FOG-2 function as negative regulators of blood, heart, and eye development in
Drosophila
Authors: Fossett Nancy , Tevosian Sergei G. , Gajewski Kathleen , Zhang Qian , Orkin Stuart H. , Schulz Robert A.
Published: July 2002

Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The
Drosophila
FOG homologue U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homologue Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. In this report, we show that Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of
ush
function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in
Drosophila
. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in
Drosophila
, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages.
A TRP homolog in
Saccharomyces cerevisiae
forms an intracellular Ca
2+
-permeable channel in the yeast vacuolar membrane
Authors: Palmer Chris P. , Zhou Xin-Liang , Lin Junyu , Loukin Stephen H. , Kung Ching , Saimi Yoshiro
Published: July 2002

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in
Saccharomyces cerevisiae
. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast
TRP
-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca
2+
activates the YVC1 current carried by cations including Ca
2+
across the vacuolar membrane. Immunodetection of a tagged
YVC1
gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the
YVC1
vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.
Act1, an NF-κB-activating protein
Authors: Li Xiaoxia , Commane Mairead , Nie Huiqin , Hua Xianxin , Chatterjee-Kishore Moitreyee , Wald David , Haag Michael , Stark George R.
Published: July 2002

Use of an NF-κB-dependent selectable marker facilitated the isolation of a cell line containing a cDNA encoding Act1, an NF-κB activator. Act1 associates with and activates IκB kinase (IKK), leading to the liberation of NF-κB from its complex with IκB. Many signaling pathways that liberate NF-κB also activate activating transcription factor (ATF) and activator protein 1 (AP-1) through Jun kinase (JNK). Act1 also activates JNK, suggesting that it might be part of a multifunctional complex involved in the activation of both NF-κB and JNK. Act1 fails to activate NF-κB in an IL-1-unresponsive mutant cell line in which all known signaling components are present, suggesting that it interacts with an unknown component in IL-1 signaling.
Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-α bigenic mice
Authors: Lenferink Anne E. G. , Simpson Jean F. , Shawver Laura K. , Coffey Robert J. , Forbes James T. , Arteaga Carlos L.
Published: July 2002

Overexpression of ErbB-2/Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)/Neu + MMTV/transforming growth factor α bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogen-activated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27
Kip1
. In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly
in vitro
by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1/2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant
in vitro
. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27
in vitro
. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.
The role of dynamic palmitoylation in Ca
2+
channel inactivation
Authors: Hurley Joyce H. , Cahill Anne L. , Currie Kevin P. M. , Fox Aaron P.
Published: July 2002

N- and P/Q-type Ca
2+
channels regulate a number of critical physiological processes including synaptic transmission and hormone secretion. These Ca
2+
channels are multisubunit proteins, consisting of a pore-forming α
1
, and accessory β and α
2
δ subunits each encoded by multiple genes and splice variants. β subunits alter current amplitude and kinetics. The β
2a
subunit is associated with slowed inactivation, an effect that requires the palmitoylation of two N-terminal cysteine residues in β
2a
. In the current manuscript, we studied steady state inactivation properties of native N- and P/Q-type Ca
2+
channels and recombinant N-type Ca
2+
channels. When bovine α
1B
and β
2a
and human α
2
δ were coexpressed in tsA 201 cells, we observed significant variations in inactivation; some cells exhibited virtually no inactivation as the holding potential was altered whereas others exhibited significant inactivation. A similar variability in inactivation was observed in native channels from bovine chromaffin cells. In individual chromaffin cells, the amount of inactivation exhibited by N-type channels was correlated with the inactivation of P/Q-type channels, suggesting a shared mechanism. Our results with recombinant channels with known β subunit composition indicated that inactivation could be dynamically regulated, possibly by alterations in β subunit palmitoylation. Tunicamycin, which inhibits palmitoylation, increased steady-state inactivation of Ca
2+
channels in chromaffin cells. Cerulenin, another drug that inhibits palmitoylation, also increased inactivation. Tunicamycin produced a similar effect on recombinant N-type Ca
2+
channels containing β
2a
but not β
2b
or β
2a
subunits mutated to be palmitoylation deficient. Our results suggest that Ca
2+
channels containing β
2a
subunits may be regulated by dynamic palmitoylation.
Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells
Authors: Loonstra Ate , Vooijs Marc , Beverloo H. Berna , Allak Bushra Al , van Drunen Ellen , Kanaar Roland , Berns Anton , Jonkers Jos
Published: July 2002

The use of Cre/
loxP
recombination in mammalian cells has expanded rapidly. We describe here that Cre expression in cultured mammalian cells may result in a markedly reduced proliferation and that this effect is dependent on the endonuclease activity of Cre. Chromosome analysis after Cre expression revealed numerous chromosomal aberrations and an increased number of sister chromatid exchanges. Titration experiments in mouse embryo fibroblasts with a ligand-regulatable Cre-ER
T
show that toxicity is dependent on the level of Cre activity. Prolonged, low levels of Cre activity permit recombination without concomitant toxicity. This urges for a careful titration of Cre activity in conditional gene modification in mammalian cells.
Transpiration, a prerequisite for long-distance transport of minerals in plants?
Authors: Tanner W. , Beevers H.
Published: July 2002

The major “benefit” alleged to accrue from transpiration (the evaporative loss of water from plant surfaces) is that it is essential for the long-distance transport of mineral ions, but the possible interrelation between these two processes has rarely been tested. Transpiration was experimentally dissociated from mineral supply by growing sunflowers (
Helianthus anuus
) in hydroculture and providing mineral nutrients only during the nights. These plants grew as well as a control group that received nutrients only during the day and transpired 12–15 times more water during the exposure period. It thus appears that convective water transport in the xylem, brought about by root pressure and the resultant guttation, “growth water,” and Münch's phloem counterflow is in itself sufficient for long-distance mineral supply and that transpiration is not required for this function.
Type I topoisomerase activity is required for proper chromosomal segregation in
Escherichia coli
Authors: Zhu Qing , Pongpech Pintip , DiGate Russell J.
Published: July 2002

Type I DNA topoisomerases are ubiquitous enzymes involved in
many aspects of DNA metabolism.
Escherichia coli
possesses two type I topoisomerase activities, DNA topoisomerase I
(Topo I) and III (Topo III). The gene encoding Topo III
(
topB
) can be deleted without affecting cell viability.
Cells possessing a deletion of the gene encoding Topo I
(
topA
) are only viable in the presence of an additional
compensatory mutation. In the presence of compensatory mutations, Topo
I deletion strains grow normally; however, if Topo III activity is
repressed in these cells, they filament extensively and possess an
abnormal nucleoid structure. These defects can be suppressed by the
deletion of the
recA
gene, suggesting that these enzymes
may be involved in RecA-mediated recombination and may specifically
resolve recombination intermediates before partitioning.
Intragenic recombination generated two distinct
Cf
genes that mediate AVR9 recognition in the natural population of
Lycopersicon
pimpinellifolium
Authors: Van der Hoorn Renier A. L. , Kruijt Marco , Roth Ronelle , Brandwagt Bas F. , Joosten Matthieu H. A. J. , De Wit Pierre J. G. M.
Published: July 2002

Resistance gene
Cf-9
of cultivated tomato (
Lycopersicon esculentum
) confers recognition of the AVR9 elicitor protein of the fungal pathogen
Cladosporium fulvum
. The
Cf-9
locus, containing
Cf-9
and four homologs (
Hcr9
s), originates from
Lycopersicon pimpinellifolium
(
Lp
). We examined naturally occurring polymorphism in
Hcr9
s that confer AVR9 recognition in the
Lp
population. AVR9 recognition occurs frequently throughout this population. In addition to
Cf-9,
we discovered a second gene in
Lp
, designated
9DC,
which also confers AVR9 recognition. Compared with
Cf-9
,
9DC
is more polymorphic, occurs more frequently, and is more widely spread throughout the
Lp
population, suggesting that
9DC
is older than
Cf-9
. The sequences of
Cf-9
and
9DC
suggest that
Cf-9
evolved from
9DC
by intragenic recombination between
9DC
and another
Hcr9.
The fact that the 9DC and Cf-9 proteins differ in 61 aa residues, and both mediate recognition of AVR9, shows that in nature Hcr9 proteins with the same recognitional specificity can vary significantly.
Quantum computing
Authors: Li Shu-Shen , Long Gui-Lu , Bai Feng-Shan , Feng Song-Lin , Zheng Hou-Zhi
Published: July 2002

Quantum computing is a quickly growing research field. This article introduces the basic concepts of quantum computing, recent developments in quantum searching, and decoherence in a possible quantum dot realization.
Microbial iron transport via a siderophore shuttle: A membrane ion transport paradigm
Authors: Stintzi Alain , Barnes Carmen , Xu Jide , Raymond Kenneth N.
Published: July 2002

A mechanism of ion transport across membranes is reported. Microbial transport of Fe3+generally delivers iron, a growth-limiting nutrient, to cells via highly specific siderophore-mediated transport systems. In contrast, iron transport in the fresh water bacteriumAeromonas hydrophilais found to occur by means of an indiscriminant siderophore transport system composed of a single multifunctional receptor. It is shown that (i) the siderophore and Fe3+enter the bacterium together, (ii) a ligand exchange step occurs in the course of the transport, and (iii) a redox process is not involved in iron exchange. To the best of our knowledge, there have been no other reports of a ligand exchange mechanism in bacterial iron transport. The ligand exchange step occurs at the cell surface and involves the exchange of iron from a ferric siderophore to an iron-free siderophore already bound to the receptor. This ligand exchange mechanism is also found inEscherichia coliand seems likely to be widely distributed among microorganisms.
DNA ligase IV-deficient cells are more resistant to ionizing radiation in the absence of Ku70: Implications for DNA double-strand break repair
Authors: Adachi Noritaka , Ishino Takayuki , Ishii Yasuyuki , Takeda Shunichi , Koyama Hideki
Published: July 2002

Vertebrate cells have evolved two major pathways for repairing DNA double-strand breaks (DSBs), homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ). To investigate the role of DNA ligase IV (Lig4) in DSB repair, we knocked out the Lig4 gene (
LIG4
) in the DT40 chicken B-lymphocyte cell line. The
LIG4
−/−
cells showed a marked sensitivity to X-rays, bleomycin, and VP-16 and were more x-ray-sensitive in G
1
than late S or G
2
/M, suggesting a critical role of Lig4 in DSB repair by NHEJ. In support of this notion, HR was not impaired in
LIG4
−/−
cells.
LIG4
−/−
cells were more x-ray-sensitive when compared with
KU70
−/−
DT40 cells, particularly at high doses. Strikingly, however, the x-ray sensitivity of
KU70
−/−
/LIG4
−/−
double-mutant cells was essentially the same as that of
KU70
−/−
cells, showing that Lig4 deficiency has no effect in the absence of Ku. These results indicate that Lig4 is exclusively required for the Ku-dependent NHEJ pathway of DSB repair and that other DNA ligases (I and III) do not substitute for this function. Our data may explain the observed severe phenotype of Lig4-deficient mice as compared with Ku-deficient mice.
Genetic characterization of the body attributed to the evangelist Luke
Authors: Vernesi Cristiano , Di Benedetto Giulietta , Caramelli David , Secchieri Erica , Simoni Lucia , Katti Emile , Malaspina Patrizia , Novelletto Andrea , Marin Vito Terribile Wiel , Barbujani Guido
Published: July 2002

Historical sources indicate that the evangelist Luke was born in Syria, died in Greece, and then his body was transferred to Constantinople, and from there to Padua, Italy. To understand whether there is any biological evidence supporting a Syrian origin of the Padua body traditionally attributed to Luke, or a replacement in Greece or Turkey, the mtDNA was extracted from two teeth and its control region was cloned and typed. The sequence determined in multiple clones is an uncommon variant of a set of alleles that are common in the Mediterranean region. We also collected and typed modern samples from Syria and Greece. By comparison with these population samples, and with samples from Anatolia that were already available in the literature, we could reject the hypothesis that the body belonged to a Greek, rather than a Syrian, individual. However, the probability of an origin in the area of modern Turkey was only insignificantly lower than the probability of a Syrian origin. The genetic evidence is therefore compatible with the possibility that the body comes from Syria, but also with its replacement in Constantinople.
Vestigial preference functions in neural networks and túngara frogs.
Authors: Phelps S. M. , Ryan M. J. , Rand A. S.
Published: July 2002

Although there is a growing interest in understanding how perceptual mechanisms influence behavioral evolution, few studies have addressed how perception itself is shaped by evolutionary forces. We used a combination of artificial neural network models and behavioral experiments to investigate how evolutionary history influenced the perceptual processes used in mate choice by female túngara frogs. We manipulated the evolutionary history of artificial neural network models and observed an emergent bias toward calls resembling known ancestral states. We then probed female túngara frogs for similar preferences, finding strong biases toward stimuli that resemble a call hypothesized for a recent ancestor. The data strongly suggest that female túngara frogs exhibit vestigial preferences for ancestral calls, and provide a general strategy for exploring the role of historical contingency in perceptual biases.
Regulation of apoptosis at cell division by p34
cdc2
phosphorylation of survivin
Authors: O'Connor Daniel S. , Grossman Douglas , Plescia Janet , Li Fengzhi , Zhang Hui , Villa Antonello , Tognin Simona , Marchisio Pier Carlo , Altieri Dario C.
Published: July 2002

The interface between apoptosis (programmed cell death) and
the cell cycle is essential to preserve homeostasis and genomic
integrity. Here, we show that survivin, an inhibitor of
apoptosis over-expressed in cancer, physically associates
with the cyclin-dependent kinase p34
cdc2
on the mitotic
apparatus, and is phosphorylated on Thr
34
by
p34
cdc2
-cyclin B1,
in vitro
and
in
vivo
. Loss of phosphorylation on Thr
34
resulted in
dissociation of a survivin-caspase-9 complex on the mitotic apparatus,
and caspase-9-dependent apoptosis of cells traversing mitosis.
These data identify survivin as a mitotic substrate of
p34
cdc2
-cyclin B1 and suggest that survivin phosphorylation
on Thr
34
may be required to preserve cell viability at cell
division. Manipulation of this pathway may facilitate the elimination
of cancer cells at mitosis.
Protein folding and unfolding in microseconds to nanoseconds by experiment and simulation
Authors: Mayor Ugo , Johnson Christopher M. , Daggett Valerie , Fersht Alan R.
Published: July 2002

The Engrailed Homeodomain protein has the highest refolding and
unfolding rate constants directly observed to date. Temperature jump
relaxation measurements gave a refolding rate constant of 37,500
s
−1
in water at 25°C, rising to 51,000 s
−1
around 42°C. The unfolding rate constant was 1,100 s
−1
in water at 25°C and 205,000 s
−1
at 63°C. The
unfolding half-life is extrapolated to be ≈7.5 ns at 100°C, which
allows real-time molecular dynamics unfolding simulations to be tested
on this system at a realistic temperature. Preliminary simulations did
indeed conform to unfolding on this time scale. Further, similar
transition states were observed in simulations at 100°C and 225°C,
suggesting that high-temperature simulations provide results applicable
to lower temperatures.
SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase
Authors: Bennett Brydon L. , Sasaki Dennis T. , Murray Brion W. , O'Leary Eoin C. , Sakata Steve T. , Xu Weiming , Leisten Jim C. , Motiwala Aparna , Pierce Steve , Satoh Yoshitaka , Bhagwat Shripad S. , Manning Anthony M. , Anderson David W.
Published: July 2002

Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (Ki= 0.19 μM). SP600125 is a reversible ATP-competitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genesCOX-2,IL-2,IFN-γ,TNF-α, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-α and inhibited anti-CD3-induced apoptosis of CD4+CD8+thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.