bluuebunny/crossref_metadata_2025 huggingface dataset:
Syntenin, a PDZ protein that binds syndecan cytoplasmic domains
Authors: Grootjans Johan J. , Zimmermann Pascale , Reekmans Gunter , Smets An , Degeest Gisèle , Dürr Joachim , David Guido
Published: July 2002

The syndecans are transmembrane proteoglycans that place
structurally heterogeneous heparan sulfate chains at the cell surface
and a highly conserved polypeptide in the cytoplasm. Their versatile
heparan sulfate moieties support various processes of molecular
recognition, signaling, and trafficking. Here we report the
identification of a protein that binds to the cytoplasmic domains of
the syndecans in yeast two-hybrid screens, surface plasmon resonance
experiments, and ligand-overlay assays. This protein, syntenin,
contains a tandem repeat of PDZ domains that reacts with the FYA
C-terminal amino acid sequence of the syndecans. Recombinant enhanced
green fluorescent protein (eGFP)–syntenin fusion proteins decorate the
plasmamembrane and intracellular vesicles, where they colocalize and
cosegregate with syndecans. Cells that overexpress eGFP–syntenin show
numerous cell surface extensions, suggesting effects of syntenin on
cytoskeleton–membrane organization. We propose that syntenin may
function as an adaptor that couples syndecans to cytoskeletal proteins
or cytosolic downstream signal-effectors.
Wild-type
Escherichia coli
grows on the chitin disaccharide,
N,N
′-diacetylchitobiose, by expressing the
cel
operon
Authors: Keyhani Nemat O. , Roseman Saul
Published: July 2002

We report here that wild-type
Escherichia coli
can grow on the chitin disaccharide,
N,N
′-diacetylchitobiose (GlcNAc)
2
, as the sole source of carbon. Transposon mutants were isolated that were unable to ferment (GlcNAc)
2
but grew normally on the monosaccharide GlcNAc. One such mutant was used to screen a wild-type
E. coli
genomic cosmid library for restoration of (GlcNAc)
2
fermentation. A partial sequence analysis of the isolated fragment mapped the clone to the (previously sequenced)
E. coli
genome between 39.0 and 39.2 min. The nucleotide ORFs at this region had been previously assigned to code for a “cryptic” cellobiose utilization (
cel
) operon. We report here, however, that functional analysis of the operon, including growth and chemotaxis, reveal that it encodes a set of proteins that are not cryptic, but are induced by (GlcNAc)
2
and catabolize the disaccharide. We therefore propose to rename the
cel
operon as the
chb
(
N,N
′-diacetyl
ch
ito
b
iose) operon, with the letter designation of the genes of the operon to be reassigned consistent with the nomenclature based on functional characterization of the gene products as follows:
celA
to
chbB
,
celB
to
chbC
,
celC
to
chbA
,
celD
to
chbR
, and
celF
to
chbF
. Furthermore, sequencing evidence indicates that the operon contains an additional gene of unknown function to be designated as
chbG
. Thus, the overall gene sequence is to be named
chbBCARFG
.
Conserved biological function betweenPax-2 andPax-5 in midbrain and cerebellum development: Evidence from targeted mutations
Authors: Schwarz Martin , Alvarez-Bolado Gonzalo , Urbánek Pavel , Busslinger Meinrad , Gruss Peter
Published: July 2002

The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer (Joyner, A. L. (1996)Trends Genet.12, 15–201). The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish (Krauss, S., Maden, M., Holder, N. & Wilson, S. W. (1992)Nature (London)360, 87–89) with neutralizing antibodies againstPax-b, the orthologue ofPax-2in mouse, placed this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation ofPax-5has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas thePax-2null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function betweenPax-2andPax-5we analyzed the brain phenotypes of mice expressing different dosages of both genes. Our results demonstrate a conserved biological function of both proteins in midbrain/hindbrain regionalization. Additionally, we show that one allele ofPax-2, but notPax-5, is necessary and sufficient for midbrain and cerebellum development in C57BL/6 mice.
In vivo
gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector
Authors: Hara Toshifumi , Tan Yadi , Huang Leaf
Published: July 2002

Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and/or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles (≈100 nm in diameter) with an efficiency ≈65%. When injected into the portal vein of mice, there were ≈5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 μg injected DNA. This level of transgene expression was ≈100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 μg DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human α1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier.
Protective anti-tumor immunity induced by vaccination with recombinant adenoviruses encoding multiple tumor-associated cytotoxic T lymphocyte epitopes in a string-of-beads fashion
Authors: Toes René E. M. , Hoeben Rob C. , van der Voort Ellen I. H. , Ressing Maaike E. , van der Eb Alex J. , Melief Cornelis J. M. , Offringa Rienk
Published: July 2002

Vaccines harboring genes that encode functional oncoproteins are intrinsically hazardous, as their application may lead to introduction of these genes into normal cells and thereby to tumorigenesis. On the other hand, oncoproteins are especially attractive targets for immunotherapy of cancer, as their expression is generally required for tumor growth, making the arisal of tumor variants lacking these antigens unlikely. Using murine tumor models, we investigated the efficacy of polyepitope recombinant adenovirus (rAd) vaccines, which encode only the immunogenic T cell epitopes derived from several oncogenes, for the induction of protective anti-tumor immunity. We chose to employ rAd, as these are safe vectors that do not induce the side effects associated with, for example, vaccinia virus vaccines. A single polyepitope rAd was shown to give rise to presentation of both H-2 and human leukocyte antigen-restricted cytotoxic T lymphocyte (CTL) epitopes. Moreover, vaccination with a rAd encoding H-2-restricted CTL epitopes, derived from human adenovirus type 5 early region 1 and human papilloma virus type 16-induced tumors, elicited strong tumor-reactive CTL and protected the vaccinated animals against an otherwise lethal challenge with either of these tumors. The protection induced was superior compared with that obtained by vaccination with irradiated tumor cells. Thus, vaccination with polyepitope rAd is a powerful approach for the induction of protective anti-tumor immunity that allows simultaneous immunization against multiple tumor-associated T cell epitopes, restricted by various major histocompatibility complex haplotypes.
Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase
Authors: Wang Yong-Xiao , Kotlikoff Michael I.
Published: July 2002

To determine the mechanisms responsible for the termination of Ca
2+
-activated Cl
−
currents (I
Cl(Ca)
), simultaneous measurements of whole cell currents and intracellular Ca
2+
concentration ([Ca
2+
]
i
) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, I
Cl(Ca)
decayed before the [Ca
2+
]
i
decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca
2+
]
i
. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of I
Cl(Ca)
, resulting in a rate of current decay similar to that of the fall in [Ca
2+
]
i
. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca
2+
/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of I
Cl(Ca)
decay was slowed and matched the [Ca
2+
]
i
decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca
2+
]
i
was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, I
Cl(Ca)
did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-
O
-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial I
Cl(Ca)
, and little or no current (despite a normal [Ca
2+
]
i
transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.
Mutations in the
mariner
transposase: The D,D(35)E consensus sequence is nonfunctional
Authors: Lohe Allan R. , De Aguiar Daniel , Hartl Daniel L.
Published: July 2002

Genetic analysis of eukaryote transposases and comparison with their prokaryote counterparts have been greatly hindered by difficulty in isolating mutations. We describe a simple eye-color screen that facilitates isolation and analysis of mutations in the
mariner
transposase in
Drosophila melanogaster
. Use of ethyl methanesulfonate and site-directed mutagenesis has identified 18 residues that are critical for
in vivo
excision of a target
mariner
element. When the mutations were examined in heterozygous mutant/nonmutant genotypes, more than half of the mutant transposase proteins were found to reduce the activity of the wild-type transposase, as assayed by the frequency of germline excision of a target element. Remarkably, transposase function is obliterated when the D,D(34)D acidic, ion-binding domain is replaced with the consensus sequence D,D(34)E found in the nematode Tc
1
transposase and in many other transposases in the superfamily. A number of mutations strongly complement wild-type transposase in a dominant-negative manner, suggestive of subunit interactions in the excision reaction; these mutations are located in a small region that includes part of the D,D(34)D motif. Transposase function also is eliminated by a mutation in the inferred initiation codon and by a mutation in a putative nuclear localization signal.
Clade B-based HIV-1 vaccines elicit cross-clade cytotoxic T lymphocyte reactivities in uninfected volunteers
Authors: Ferrari Guido , Humphrey Will , McElrath M. Juliana , Excler Jean–Louis , Duliege Anne–Marie , Clements Mary Lou , Corey Lawrence C. , Bolognesi Dani P. , Weinhold Kent J.
Published: July 2002

A fundamental goal of current strategies to develop an efficacious vaccine for AIDS is the elicitation of broadly reactive cytotoxic T lymphocyte (CTL) reactivities capable of destroying virally infected targets. Recent application of recombinant canarypox ALVAC/HIV-1 vectors as vaccine immunogens in HIV-1,-noninfected volunteers has produced CTL responses in a significant number of vaccinees. Using a newly developed targeting strategy, we examined the capacity of vaccine-induced CTL to lyse autologous targets infected with a diverse group of viral isolates. CTL derived from recipients of a canarypox ALVAC/HIV-1 gp160 (MN) vaccine were found capable of lysing autologous CD4+lymphoblasts infected with the prototypic LAI strain of HIV-1. When tested against autologous targets infected with primary HIV-1 isolates representing genetically diverse viral clades, CTL from ALVAC/gp160 recipients showed both a broad pattern of cytolysis in which viruses from all clades tested were recognized as well as a highly restricted pattern in which no primary isolates, including clade B, were lysed. Differences in the HLA haplotypes of the volunteers immunized with the envelope vector might be a major determinant of the relative breadth of their CTL response. In contrast to ALVAC/gp160 vaccinees, recipients of the ALVAC/HIV-1 immunogen containing envelope as well as gag and protease genes consistently had CTL reactivities effective against a spectrum of primary isolate-infected targets. These studies demonstrate for the first time that clade B-based canarypox vaccines can elicit broad CTL reactivities capable of recognizing viruses belonging to genetically diverse HIV-1 clades. The results also reinforce the impact of viral core elements in the vaccine as well as the pattern of major histocompatibility complex class I allelic expression by the vaccine recipient in determining the relative breadth of the cellular response.
Voltage-dependent gating characteristics of the K
+
channel KAT1 depend on the N and C termini
Authors: Marten Irene , Hoshi Toshinori
Published: July 2002

We studied how the C and N termini of the plant K
+
channel KAT1 influence the voltage-dependent gating behavior by generating C- and N-terminal deletion mutants. Functional expression was observed only when C-terminal deletions were downstream of the putative cyclic nucleotide binding site. Treatments of oocytes expressing KAT1 channels with anticytoskeletal agents indicated that intact microtubules are important for functional expression. C-terminal deletions altered the voltage sensitivity of the KAT1 channel with greater deletions resulting in smaller equivalent charge movements. In contrast, a deletion in the N terminus (Δ20–34) shifted the half-activation voltage by approximately −65 mV without markedly affecting the number of equivalent charges. The results reveal novel roles of the N and C termini in regulation of the voltage-dependent gating of KAT1.
Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: Prosthetic group characterization and cDNA cloning
Authors: Rathinasabapathi Bala , Burnet Michael , Russell Brenda L. , Gage Douglas A. , Liao Pao-Chi , Nye Gordon J. , Scott Paul , Golbeck John H. , Hanson Andrew D.
Published: July 2002

Plants synthesize the osmoprotectant glycine betaine via the route choline → betaine aldehyde → glycine betaine. In spinach, the first step is catalyzed by choline monooxygenase (CMO), a ferredoxin-dependent stromal enzyme that has been hypothesized to be an oligomer of identical subunits and to be an Fe-S protein. Analysis by HPLC and matrix-assisted laser desorption ionization MS confirmed that native CMO contains only one type of subunit (
M
r
42,864). Determination of acid-labile sulfur and nonheme iron demonstrated that there is one [2Fe-2S] cluster per subunit, and EPR spectral data indicated that this cluster is of the Rieske type—i.e., coordinated by two Cys and two His ligands. A full-length CMO cDNA (1,622 bp) was cloned from spinach using a probe generated by PCR amplification for which the primers were based on internal peptide sequences. The ORF encoded a 440-amino acid polypeptide that included a 60-residue transit peptide. The deduced amino acid sequence included two Cys-His pairs spaced 16 residues apart, a motif characteristic of Rieske-type Fe-S proteins. Larger regions that included this motif also showed some sequence similarity (≈40%) to Rieske-type proteins, particularly bacterial oxygenases. Otherwise there was very little similarity between CMO and proteins from plants or other organisms. RNA and immunoblot analyses showed that the expression of CMO in leaves increased several-fold during salinization. We conclude that CMO is a stress-inducible representative of a new class of plant oxygenases.
Isolation of a hepadnavirus from the woolly monkey, a New World primate
Authors: Lanford Robert E. , Chavez Deborah , Brasky Kathleen M. , Burns Roy B. , Rico-Hesse Rebeca
Published: July 2002

Hepatitis B virus (HBV) infections are a major worldwide health problem with chronic infections leading to cirrhosis and liver cancer. Viruses related to human HBV have been isolated from birds and rodents, but despite efforts to find hepadnaviruses that infect species intermediate in evolution between rodents and humans, none have been described. We recently isolated a hepadnavirus from a woolly monkey (
Lagothrix lagotricha
) that was suffering from fulminant hepatitis. Phylogenetic analysis of the nucleotide sequences of the core and surface genes indicated that the virus was distinct from the human HBV family, and because it is basal (ancestral) to the human monophyletic group, it probably represents a progenitor of the human viruses. This virus was designated woolly monkey hepatitis B virus (WMHBV). Analysis of woolly monkey colonies at five zoos indicated that WMHBV infections occurred in most of the animals at the Louisville zoo but not at four other zoos in the United States. The host range of WMHBV was examined by inoculation of one chimpanzee and two black-handed spider monkeys (
Ateles geoffroyi
), the closest nonendangered relative of the woolly monkey. The data suggest that spider monkeys are susceptible to infection with WMHBV and that minimal replication was observed in a chimpanzee. Thus, we have isolated a hepadnavirus with a host intermediate between humans and rodents and establishes a new animal model for evaluation of antiviral therapies for treating HBV chronic infections.
Molecular characterization of the phenylacetic acid catabolic pathway in
Pseudomonas putida
U: The phenylacetyl-CoA catabolon
Authors: Olivera E. R. , Miñambres B. , García B. , Muñiz C. , Moreno M. A. , Ferrández A. , Díaz E. , García J. L. , Luengo J. M.
Published: July 2002

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by
Pseudomonas putida
U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (
i
) a transport system, (
ii
) a phenylacetic acid activating enzyme, (
iii
) a ring-hydroxylation complex, (
iv
) a ring-opening protein, (
v
) a β-oxidation-like system, and (
vi
) two regulatory genes. This pathway constitutes the common part (
core
) of a complex functional unit (
catabolon
) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).
Thegastrulation defectivegene ofDrosophila melanogasteris a member of the serine protease superfamily
Authors: Konrad Kenneth D. , Goralski Thomas J. , Mahowald Anthony P. , Marsh J. Lawrence
Published: July 2002

The establishment of dorsal–ventral polarity in the oocyte involves two sets of genes. One set belongs to thegurken-torpedosignaling pathway and affects the development of the egg chorion as well as the polarity of the embryo. The second set of genes affects only the dorsal–ventral polarity of the embryo but not the eggshell.gastrulation defectiveis one of the earliest acting of this second set of maternally required genes. We have cloned and characterized thegastrulation defectivegene and determined that it encodes a protein structurally related to the serine protease superfamily, which also includes the Snake, Easter, and Nudel proteins. These data provide additional support for the involvement of a protease cascade in generating an asymmetric signal (i.e., asymmetric Spätzle activity) during establishment of dorsal–ventral polarity in theDrosophilaembryo.
Synchronization between prefrontal and posterior association cortex during human working memory
Authors: Sarnthein J. , Petsche H. , Rappelsberger P. , Shaw G. L. , von Stein A.
Published: July 2002

We measured coherence between the electroencephalogram at different scalp sites while human subjects performed delayed response tasks. The tasks required the retention of either verbalizable strings of characters or abstract line drawings. In both types of tasks, a significant enhancement in coherence in the θ range (4–7 Hz) was found between prefrontal and posterior electrodes during 4-s retention intervals. During 6-s perception intervals, far fewer increases in θ coherence were found. Also in other frequency bands, coherence increased; however, the patterns of enhancement made a relevance for working memory processes seem unlikely. Our results suggest that working memory involves synchronization between prefrontal and posterior association cortex by phase-locked, low frequency (4–7 Hz) brain activity.
Structural, functional, and evolutionary relationships between λ-exonuclease and the type II restriction endonucleases
Authors: Kovall Rhett A. , Matthews Brian W.
Published: July 2002

λ-exonuclease participates in DNA recombination and repair. It binds a free end of double-stranded DNA and degrades one strand in the 5′ to 3′ direction. The primary sequence does not appear to be related to any other protein, but the crystal structure shows part of λ-exonuclease to be similar to the type II restriction endonucleases
Pvu
II and
Eco
RV. There is also a weaker correspondence with
Eco
RI,
Bam
HI, and Cfr10I. The structure comparisons not only suggest that these enzymes all share a similar catalytic mechanism and a common structural ancestor but also provide strong evidence that the toroidal structure of λ-exonuclease encircles its DNA substrate during hydrolysis.
PRT1
of
Arabidopsis thaliana
encodes a component of the plant N-end rule pathway
Authors: Potuschak Thomas , Stary Susanne , Schlögelhofer Peter , Becker Frank , Nejinskaia Victoria , Bachmair Andreas
Published: July 2002

Mutants in the
PRT1
gene of
Arabidopsis thaliana
are impaired in the degradation of a normally short-lived intracellular protein that contains a destabilizing N-terminal residue. Proteins bearing such residues are the substrates of an ubiquitin-dependent proteolytic system called the N-end rule pathway. The chromosomal position of
PRT1
was determined, and the
PRT1
gene was isolated by map-based cloning. The 45-kDa
PRT1
protein contains two RING finger domains and one ZZ domain. No other proteins in databases match these characteristics of
PRT1
. There is, however, a weak similarity to Rad18p of
Saccharomyces cerevisiae
. The RING finger domains have been found in a number of other proteins that are involved in ubiquitin conjugation, consistent with the proposed role of
PRT1
in the plant N-end rule pathway.
Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America
Authors: Berger Lee , Speare Rick , Daszak Peter , Green D. Earl , Cunningham Andrew A. , Goggin C. Louise , Slocombe Ron , Ragan Mark A. , Hyatt Alex D. , McDonald Keith R. , Hines Harry B. , Lips Karen R. , Marantelli Gerry , Parkes Helen
Published: July 2002

Epidermal changes caused by a chytridiomycete fungus (Chytridiomycota; Chytridiales) were found in sick and dead adult anurans collected from montane rain forests in Queensland (Australia) and Panama during mass mortality events associated with significant population declines. We also have found this new disease associated with morbidity and mortality in wild and captive anurans from additional locations in Australia and Central America. This is the first report of parasitism of a vertebrate by a member of the phylum Chytridiomycota. Experimental data support the conclusion that cutaneous chytridiomycosis is a fatal disease of anurans, and we hypothesize that it is the proximate cause of these recent amphibian declines.
Protein
trans
-splicing by a split intein encoded in a split DnaE gene of
Synechocystis
sp. PCC6803
Authors: Wu Hong , Hu Zhuma , Liu Xiang-Qin
Published: July 2002

A split intein capable of protein
trans
-splicing is identified in a DnaE protein of the cyanobacterium
Synechocystis
sp. strain PCC6803. The N- and C-terminal halves of DnaE (catalytic subunit α of DNA polymerase III) are encoded by two separate genes,
dnaE-n
and
dnaE-c
, respectively. These two genes are located 745,226 bp apart in the genome and on opposite DNA strands. The
dnaE-n
product consists of a N-extein sequence followed by a 123-aa intein sequence, whereas the
dnaE-c
product consists of a 36-aa intein sequence followed by a C-extein sequence. The N- and C-extein sequences together reconstitute a complete DnaE sequence that is interrupted by the intein sequences inside the β- and τ-binding domains. The two intein sequences together reconstitute a split mini-intein that not only has intein-like sequence features but also exhibited protein
trans
-splicing activity when tested in
Escherichia coli
cells.
Bursting calcium rotors in cultured cardiac myocyte monolayers
Authors: Bub Gil , Glass Leon , Publicover Nelson G. , Shrier Alvin
Published: July 2002

Rotating waves (rotors) of cellular activity were observed in nonconfluent cultures of embryonic chick heart cells by using a macroscopic imaging system that detected fluorescence from intracellular Ca2+. Unlike previous observations of rotors or spiral waves in other systems, the rotors did not persist but exhibited a repetitive pattern of spontaneous onset and offset leading to a bursting rhythm. Similar dynamics were observed in a cellular automaton model of excitable media that incorporates spontaneous initiation of activity, and a decrease of excitability as a consequence of rapid activity (fatigue). These results provide a mechanism for bursting dynamics in normal and pathological biological processes.
Random mutagenesis of 1-aminocyclopropane-1-carboxylate synthase: A key enzyme in ethylene biosynthesis
Authors: Tarun Alice S. , Lee Joon S. , Theologis Athanasios
Published: July 2002

1-Aminocyclopropane-1-carboxylate synthase (ACC synthase, EC
4.4.1.14
) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. To determine the amino acid residues critical for the structure and function of this enzyme, the tomato Le-ACS2 isoenzyme has been subjected to both site-directed and PCR random mutagenesis. Mutant ACC synthases with reduced enzyme activity have been selected by using a genetic screen based on the functional complementation of an
Escherichia coli
Ile auxotroph that has been engineered to express ACC deaminase from
Pseudomonas
sp. The DNA sequence of almost 1,000 clones has been determined, and 334 single missense mutations have been selected for analysis. We have identified three classes of mutants based on their activity and expression in
E. coli.
Class I and II mutants have the same level of protein expression as the wild type, but their enzyme activity is reduced to 0–5% and 5–50%, respectively. Class III mutants have neither activity nor detectable protein expression. The inactive mutations are clustered in regions that are highly conserved among various ACC synthases. This library of mutants will facilitate the elucidation of structure-function relationships of this regulatory enzyme.
Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on
Escherichia coli
K12
Authors: Ralph Edward T. , Guest John R. , Green Jeffrey
Published: July 2002

The recent outbreaks of
Escherichia coli
0157-associated food poisoning have focused attention on the virulence determinants of
E. coli
. Here, it is reported that single base substitutions in the
fnr
gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on
E. coli
K12, the widely used laboratory strain. The mechanism involves enhancing the expression of a normally dormant hemolysin gene (
hlyE
) located in the
E. coli
chromosome. The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase. It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of
E. coli
in nature.
Expression of a dominant negative TrkB receptor, T1, reveals a requirement for presynaptic signaling in BDNF-induced synaptic potentiation in cultured hippocampal neurons
Authors: Li Yong-Xin , Xu Youfeng , Ju Donghong , Lester Henry A. , Davidson Norman , Schuman Erin M.
Published: July 2002

We have developed a method to analyze the relative contributions of pre- and postsynaptic actions of a particular gene product in neurons in culture and potentially in slices using adenovirus-mediated gene transfer. A recombinant virus directed the expression of both a GFP reporter protein and TrkB.T1, a C-terminal truncated dominant negative TrkB neurotrophin receptor. When expressed in the presynaptic cell at synapses between embryonic hippocampal neurons in culture, the dominant negative TrkB.T1 inhibited two forms of synaptic potentiation induced by the neurotrophin brain-derived neurotrophic factor (BDNF): (i) greater evoked synaptic transmission and (ii) higher frequency of spontaneous miniature synaptic currents. These inhibition effects are not seen if the transgene is expressed only in the postsynaptic cell. We conclude that BDNF-TrkB signal transduction in the presynaptic terminal leads to both types of potentiation and is therefore the primary cause of synaptic enhancement by BDNF in these neurons.
Ciona intestinalis
nuclear receptor 1: A member of steroid/thyroid hormone receptor family
Authors: Carosa Eleonora , Fanelli Albertina , Ulisse Salvatore , Di Lauro Roberto , Rall J. Edward , Jannini Emmanuele A.
Published: July 2002

Nuclear hormone receptors comprise a large family of zinc finger transcription factors, some with hydrophobic ligands, such as thyroid hormone, vitamin D, steroids, etc., and others for which no ligand has been found. Thyroid hormone receptors (TRs) generally are considered to be confined to the vertebrata that possess a thyroid gland. Tunicates represent the most primitive of the chordates, and there are data supporting a role for thyroid hormone in their metamorphosis, but no data are available on TRs in this genus; hence, we have studied
Ciona intestinalis.
Screening of a
Ciona
library with the DNA binding domain of
Xenopus laevis
TR (xTR) resulted in the isolation of a nuclear hormone receptor,
C. intestinalis
nuclear receptor 1 (CiNR1). CiNR1 is similar to TRs of more evolved species with a conserved DNA binding domain whereas the ligand binding domain shows poor homology to vertebrate sequences. The C-terminal part of CiNR1 spans ≈200 amino acids more than other TRs, lacks the AF2 transactivation domain, and is not able to bind triiodothyronine. Phylogenetically, CiNR1 appears to be close to the common ancestral gene of TRs. Expression of CiNR1 was limited to the developing embryo and the larval stage, which suggests a role during development and metamorphosis. In transfection experiments, CiNR1 down-regulated basal transcription of a reporter gene driven by the TR palindrome responsive element. When CiNR1 was cotransfected with chicken TRα, it attenuated the normal thyroid hormone response in a dominant negative fashion. This attenuation required the C-terminal portion of the molecule.
The absence of phloem loading in willow leaves
Authors: Turgeon Robert , Medville Richard
Published: July 2002

Willow (
Salix babylonica
L.) is representative of a large group of plants that have extensive plasmodesmatal connections between minor vein phloem and adjoining cells. Because plasmodesmata provide a diffusion pathway for small molecules, it is unclear how sucrose could be loaded from the mesophyll into the phloem against a concentration gradient. In the studies reported here, the minor vein phloem of willow leaves plasmolyzed in approximately the same concentration of osmoticum as the mesophyll. Sucrose concentrations in mesophyll cells were greater than those reported in the literature for aphid stylet exudate from willow stems. Calculated turgor pressures in the mesophyll and minor vein phloem were greater than turgor reported in the literature for sieve elements in the stems of willow. Images of minor veins were not obtained in autoradiographs when attached leaves, or leaf pieces, were provided with
14
CO
2
or [
14
C]sucrose. Therefore, no evidence could be found for accumulation of sucrose against a concentration gradient in the minor vein phloem of willow. In these leaves, the mesophyll apparently acts as the “source” for long distance transport of sugar. The mechanism of translocation in willow, and the evolution of phloem loading, are discussed.
Riboregulation in
Escherichia coli
: DsrA RNA acts by RNA:RNA interactions at multiple loci
Authors: Lease Richard A. , Cusick Michael E. , Belfort Marlene
Published: July 2002

DsrA is an 87-nt untranslated RNA that regulates both the global transcriptional silencer and nucleoid protein H-NS and the stationary phase and stress response sigma factor RpoS (σ
s
). We demonstrate that DsrA acts via specific RNA:RNA base pairing interactions at the
hns
locus to antagonize H-NS translation. We also give evidence that supports a role for RNA:RNA interactions at the
rpoS
locus to enhance RpoS translation. Negative regulation of
hns
by DsrA is achieved by the RNA:RNA interaction blocking translation of
hns
RNA. In contrast, results suggest that positive regulation of
rpoS
by DsrA occurs by formation of an RNA structure that activates a
cis
-acting translational operator. Sequences within DsrA complementary to three additional genes,
argR
,
ilvIH
, and
rbsD
, suggest that DsrA is a riboregulator of gene expression that acts coordinately via RNA:RNA interactions at multiple loci.
Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in
Arabidopsis
mutants
Authors: Pogson Barry J. , Niyogi Krishna K. , Björkman Olle , DellaPenna Dean
Published: July 2002

Collectively, the xanthophyll class of carotenoids perform a
variety of critical roles in light harvesting antenna assembly and
function. The xanthophyll composition of higher plant photosystems
(lutein, violaxanthin, and neoxanthin) is remarkably conserved,
suggesting important functional roles for each. We have taken a
molecular genetic approach in
Arabidopsis
toward defining
the respective roles of individual xanthophylls
in vivo
by using a series of mutant lines that selectively eliminate and
substitute a range of xanthophylls
.
The mutations,
lut1
and
lut2
(
lut
=
lutein deficient), disrupt lutein biosynthesis. In
lut2,
lutein is replaced mainly by a stoichiometric increase in violaxanthin
and antheraxanthin. A third mutant,
aba1,
accumulates
normal levels of lutein and substitutes zeaxanthin for violaxanthin and
neoxanthin. The
lut2aba1
double mutant completely lacks
lutein, violaxanthin, and neoxanthin and instead accumulates
zeaxanthin. All mutants were viable in soil and had chlorophyll
a
/
b
ratios ranging from 2.9 to 3.5 and
near wild-type rates of photosynthesis. However, mutants accumulating
zeaxanthin exhibited a delayed greening virescent phenotype, which was
most severe and often lethal when zeaxanthin was the only xanthophyll
present. Chlorophyll fluorescence quenching kinetics indicated that
both zeaxanthin and lutein contribute to nonphotochemical quenching;
specifically, lutein contributes, directly or indirectly, to the rapid
rise of nonphotochemical quenching. The results suggest that the normal
complement of xanthophylls, while not essential, is required for
optimal assembly and function of the light harvesting antenna in higher
plants.
A heterodimeric DNA polymerase: Evidence that members of Euryarchaeota possess a distinct DNA polymerase
Authors: Cann Isaac K. O. , Komori Kayoko , Toh Hiroyuki , Kanai Satoru , Ishino Yoshizumi
Published: July 2002

We describe here a DNA polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA polymerase δ, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA polymerase II (DP1 + DP2) from
Pyrococcus furiosus
, implicate the DNA polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.
Targeted expression of
teashirt
induces ectopic eyes in
Drosophila
Authors: Pan Duojia , Rubin Gerald M.
Published: July 2002

teashirt
was initially identified as a gene required for the specification of the trunk segments in
Drosophila
embryogenesis and encodes a transcription factor with zinc finger motifs. We report here that targeted expression of
teashirt
in imaginal discs is sufficient to induce ectopic eye formation in non-eye tissues, a phenotype similar to that produced from targeted expression of
eyeless
,
dachshund
, and
eyes absent
. Furthermore,
teashirt
and
eyeless
induce the expression of each other, suggesting that
teashirt
is part of the gene network that functions to specify eye identity.
Immunization against rabies with plant-derived antigen
Authors: Modelska Anna , Dietzschold Bernard , Sleysh N. , Fu Zhen Fang , Steplewski Klaudia , Hooper D. Craig , Koprowski Hilary , Yusibov Vidadi
Published: July 2002

We previously demonstrated that recombinant plant virus particles containing a chimeric peptide representing two rabies virus epitopes stimulate virus neutralizing antibody synthesis in immunized mice. We show here that mice immunized intraperitoneally or orally (by gastric intubation or by feeding on virus-infected spinach leaves) with engineered plant virus particles containing rabies antigen mount a local and systemic immune response. After the third dose of antigen, given intraperitoneally, 40% of the mice were protected against challenge infection with a lethal dose of rabies virus. Oral administration of the antigen stimulated serum IgG and IgA synthesis and ameliorated the clinical signs caused by intranasal infection with an attenuated rabies virus strain.
A third distinct tumor necrosis factor receptor of orthopoxviruses
Authors: Loparev Vladimir N. , Parsons Joseph M. , Knight Janice C. , Panus Joanne Fanelli , Ray Caroline A. , Buller R. Mark L. , Pickup David J. , Esposito Joseph J.
Published: July 2002

Cowpox virus Brighton red strain (CPV) contains a gene,
crmD
, which encodes a 320-aa tumor necrosis factor receptor (TNFR) of 44% and 22% identity, respectively, to the CPV TNFR-like proteins, cytokine response modifiers (crm) CrmB and CrmC. The
crmD
gene was interrupted in three other cowpox strains examined and absent in various other orthopoxviruses; however, four strains of ectromelia virus (ECT) examined contained an intact
crmD
(97% identity to CPV
crmD
) and lacked cognates of
crmB
and
crmC
. The protein, CrmD, contains a transport signal; a 151-aa cysteine-rich region with 21 cysteines that align with human TNFRII ligand-binding region cysteines; and C-terminal region sequences that are highly diverged from cellular TNFR C-terminal region sequences involved in signal transduction. Bacterial maltose-binding proteins containing the CPV or ECT CrmD cysteine-rich region bound TNF and lymphotoxin-α (LTα) and blocked their
in vitro
cytolytic activity. Secreted viral CrmD bound TNF and LTα and was detectable after the early stage of replication, using nonreducing conditions, as 60- to 70-kDa predominant and 90- to 250-kDa minor disulfide-linked complexes that were able to be reduced to a 46-kDa form and deglycosylated to a 38-kDa protein. Cells infected with CPV produced extremely low amounts of CrmD compared with ECT. Possessing up to three TNFRs, including CrmD, which is secreted as disulfide-linked complexes in varied amounts by CPV and ECT, likely enhances the dynamics of the immune modulating mechanisms of orthopoxviruses.
NF-κB-inducing kinase activates IKK-α by phosphorylation of Ser-176
Authors: Ling Lei , Cao Zhaodan , Goeddel David V.
Published: July 2002

Activation of the transcription factor NF-κB by inflammatory cytokines involves the successive action of NF-κB-inducing kinase (NIK) and two IκB kinases, IKK-α and IKK-β. Here we show that NIK preferentially phosphorylates IKK-α over IKK-β, leading to the activation of IKK-α kinase activity. This phosphorylation of IKK-α occurs specifically on Ser-176 in the activation loop between kinase subdomains VII and VIII. A mutant form of IKK-α containing alanine at residue 176 cannot be phosphorylated or activated by NIK and acts as a dominant negative inhibitor of interleukin 1- and tumor necrosis factor-induced NF-κB activation. Conversely, a mutant form of IKK-α containing glutamic acid at residue 176 is constitutively active. Thus, the phosphorylation of IKK-α on Ser-176 by NIK may be required for cytokine-mediated NF-κB activation.
Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2α kinase homolog
Authors: Dever Thomas E. , Sripriya Rajaraman , McLachlin Jeanne R. , Lu Jingfang , Fabian John R. , Kimball Scot R. , Miller Lois K.
Published: July 2002

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2α kinases. Insect cells infected with wild-type, but not
pk2
-deleted, baculovirus exhibited reduced eIF2α phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2α kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation
in vivo
, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.
The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast
Saccharomyces cerevisiae
Authors: Berset Catherine , Trachsel Hans , Altmann Michael
Published: July 2002

Initiation factor eIF4G is an essential protein required for initiation of mRNA translation via the 5′ cap-dependent pathway. It interacts with eIF4E (the mRNA 5′ cap-binding protein) and serves as an anchor for the assembly of further initiation factors. With treatment of
Saccharomyces cerevisiae
with rapamycin or with entry of cells into the diauxic phase, eIF4G is rapidly degraded, whereas initiation factors eIF4E and eIF4A remain stable. We propose that nutritional deprivation or interruption of the TOR signal transduction pathway induces eIF4G degradation.
Cyclin B2-null mice develop normally and are fertile whereas cyclin B1-null mice die
in utero
Authors: Brandeis Michael , Rosewell Ian , Carrington Mark , Crompton Tessa , Jacobs Mary Ann , Kirk Jane , Gannon Julian , Hunt Tim
Published: July 2002

Two B-type cyclins, B1 and B2, have been identified in mammals. Proliferating cells express both cyclins, which bind to and activate p34
cdc2
. To test whether the two B-type cyclins have distinct roles, we generated lines of transgenic mice, one lacking cyclin B1 and the other lacking cyclin B2. Cyclin B1 proved to be an essential gene; no homozygous B1-null pups were born. In contrast, nullizygous B2 mice developed normally and did not display any obvious abnormalities. Both male and female cyclin B2-null mice were fertile, which was unexpected in view of the high levels and distinct patterns of expression of cyclin B2 during spermatogenesis. We show that the expression of cyclin B1 overlaps the expression of cyclin B2 in the mature testis, but not vice versa. Cyclin B1 can be found both on intracellular membranes and free in the cytoplasm, in contrast to cyclin B2, which is membrane-associated. These observations suggest that cyclin B1 may compensate for the loss of cyclin B2 in the mutant mice, and implies that cyclin B1 is capable of targeting the p34
cdc2
kinase to the essential substrates of cyclin B2.
Toward a cancer therapy with boron-rich oligomeric phosphate diesters that target the cell nucleus
Authors: Nakanishi Akira , Guan Lufeng , Kane Robert R. , Kasamatsu Harumi , Hawthorne M. Frederick
Published: July 2002

The viability of boron neutron capture therapy depends on the development of tumor-targeting agents that contain large numbers of boron-10 (
10
B) atoms and are readily taken up by cells. Here we report on the selective uptake of homogeneous fluorescein-labeled
nido
-carboranyl oligomeric phosphate diesters (
nido
-OPDs) by the cell nucleus and their long-term retention after their delivery into the cytoplasm of TC7 cells by microinjection. All
nido
-OPDs accumulated in the cell nucleus within 2 h after microinjection. However,
nido
-OPDs in which the carborane cage was located on a side chain attached to the oligomeric backbone were redistributed between both the cytoplasm and nucleus after 24 h of incubation, whereas
nido
-OPDs in which the carborane cage was located along the oligomeric backbone remained primarily in the nucleus. Furthermore, cell-free incubation of digitonin-permeabilized TC7 cells with the
nido
-OPDs resulted in nuclear accumulation of the compounds, thus corroborating the microinjection studies. Our observation of fluorescence primarily located in the cell nucleus indicates that nuclear-specific uptake of sufficient amounts of
10
B for effective boron neutron capture therapy (≈10
8
–10
9
10
B atoms/tumor cell) via
nido
-OPDs is achievable.