bluuebunny/crossref_metadata_2025 huggingface dataset:
15-Deoxy-Δ
12,14
-prostaglandin J
2
: The endogenous electrophile that induces neuronal apoptosis
Authors: Kondo Mitsuhiro , Shibata Takahiro , Kumagai Takeshi , Osawa Toshihiko , Shibata Noriyuki , Kobayashi Makio , Sasaki Shoichi , Iwata Makoto , Noguchi Noriko , Uchida Koji
Published: July 2002

Prostaglandin D
2
(PGD
2
), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J
2
-series, such as 15-deoxy-Δ
12,14
-PGJ
2
(15d-PGJ
2
). The observation that the level of 15d-PGJ
2
increased in the tissue cells from patients with sporadic amyotrophic lateral sclerosis suggested that the formation of 15d-PGJ
2
may be closely associated with neuronal cell death during chronic inflammatory processes.
In vitro
experiments using SH-SY5Y human neuroblastoma cells revealed that 15d-PGJ
2
induced apoptotic cell death. An oligonucleotide microarray analysis demonstrated that, in addition to the heat shock-responsive and redox-responsive genes, the p53-responsive genes, such as
gadd45
,
cyclin G1
, and
cathepsin D
, were significantly up-regulated in the cells treated with 15d-PGJ
2
. Indeed, the 15d-PGJ
2
induced accumulation and phosphorylation of p53, which was accompanied by a preferential redistribution of the p53 protein in the nuclei of the cells and by a time-dependent increase in p53 DNA binding activity, suggesting that p53 accumulated in response to the treatment with 15d-PGJ
2
was functional. The 15d-PGJ
2
-induced accumulation of p53 resulted in the activation of a death-inducing caspase cascade mediated by Fas and the Fas ligand.
Long-term and homogeneous regulation of the
Escherichia coli araBAD
promoter by use of a lactose transporter of relaxed specificity
Authors: Morgan-Kiss Rachael M. , Wadler Caryn , Cronan John E.
Published: July 2002

Expression systems based on the
Escherichia coli
arabinose operon
P

BAD

promoter exhibit the all-or-nothing (autocatalytic) induction of expression that was first documented in the
lac
operon. Under conditions of subsaturating levels of inducer, some of the cells of the population are fully induced, whereas other cells remain uninduced. Recently, a new AraE transporter system was reported to have circumvented the problem of autocatalytic expression in the pBAD expression vectors and to provide graded and homogeneous cell-to-cell expression in the presence of variable inducer concentrations [Khlebnikov, A., Risa, O., Skaug, T., Carrier, T. A. & Keasling, J. D. (2000)
J. Bacteriol.
182, 7029–7034]. However, we report that nonuniform gene expression in the AraE system was readily detectable by the use of mutant green fluorescent proteins that are rapidly degraded in
E. coli
. We report an approach to avoid all-or-nothing induction of the pBAD promoter; the use of a mutant LacY transporter in a strain deficient in both arabinose transport (
araE araFGH
) and degradation (
araBAD
). This mutant LacY protein performs facilitated diffusion of arabinose resulting in homogeneous expression of an unstable GFP that is maintained over extended incubation times at subsaturating levels of inducer. This approach is readily adapted to other sugar-regulated expression systems.
Local circuit neurons in the striatum regulate neural and behavioral responses to dopaminergic stimulation
Authors: Saka E. , Iadarola M. , Fitzgerald D. J. , Graybiel A. M.
Published: July 2002

Interneurons are critical for shaping neuronal circuit activity in many parts of the central nervous system. To study interneuron function in the basal ganglia, we tested and characterized an NK-1 receptor-based method for targeted ablation of specific classes of interneuron in the striatum. Our findings demonstrate that the neurotoxin SP-PE35, a substance P–
Pseudomonas
exotoxin conjugate, selectively targets striatal cholinergic and nitric oxide synthase/somatostatinergic interneurons when injected locally into the striatum. The effects of this selective cell targeting encompassed alterations in both behavioral and neural responses to dopaminergic stimulation, including altered patterns of early-gene response in striosomes and matrix. We conclude that NK-1-bearing local circuit neurons of the striatum regulate the differential responses of striatal projection neurons to dopamine-mediated signaling.
Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa
Authors: Kedzierski Wojciech , Nusinowitz Steven , Birch David , Clarke Geoff , McInnes Roderick R. , Bok Dean , Travis Gabriel H.
Published: July 2002

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including
RDS
.
RDS
encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to
RDS
, no mutations in
ROM1
alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in
RDS
causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in
ROM1
. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with
RDS
-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in
RDS
-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.
Synaptic regulation of protein synthesis and the fragile X protein
Authors: Greenough William T. , Klintsova Anna Y. , Irwin Scott A. , Galvez Roberto , Bates Kathy E. , Weiler Ivan Jeanne
Published: July 2002

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use “synaptoneurosomes,” a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.
A chromatin insulator protects retrovirus vectors from chromosomal position effects
Authors: Emery David W. , Yannaki Evangelia , Tubb Julie , Stamatoyannopoulos George
Published: July 2002

Recombinant murine retroviruses are widely used as delivery vectors for gene therapy. However, once integrated into a chromosome, these vectors often suffer from profound position effects, with vector silencing observed
in vitro
and
in vivo
. To overcome this problem, we investigated whether the HS4 chromatin insulator from the chicken β-globin locus control region could protect a retrovirus vector from position effects. When used to flank a reporter vector, this element significantly increased the fraction of transduced cells that expressed the provirus in cultures and in mice transplanted with transduced marrow. These results demonstrate that a chromatin insulator can improve the expression performance of a widely used class of gene therapy vectors by protecting these vectors from chromosomal position effects.
Comparing sequenced segments of the tomato and
Arabidopsis
genomes: Large-scale duplication followed by selective gene loss creates a network of synteny
Authors: Ku Hsin-Mei , Vision Todd , Liu Jiping , Tanksley Steven D.
Published: July 2002

A 105-kilobase bacterial artificial chromosome (BAC) clone from the
ovate-
containing region of tomato chromosome 2 was sequenced and annotated. The tomato BAC sequence was then compared, gene by gene, with the sequenced portions of the
Arabidopsis thaliana
genome. Rather than matching a single portion of the
Arabidopsis
genome, the tomato clone shows conservation of gene content and order with four different segments of
Arabidopsis
chromosomes 2–5. The gene order and content of these individual
Arabidopsis
segments indicate that they derived from a common ancestral segment through two or more rounds of large-scale genome duplication events—possibly polyploidy. One of these duplication events is ancient and may predate the divergence of the
Arabidopsis
and tomato lineages. The other is more recent and is estimated to have occurred after the divergence of tomato and
Arabidopsis
≈112 million years ago. Together, these data suggest that, on the scale of BAC-sized segments of DNA, chromosomal rearrangements (e.g., inversions and translocations) have been only a minor factor in the divergence of genome organization among plants. Rather, the dominating factors have been repeated rounds of large-scale genome duplication followed by selective gene loss. We hypothesize that these processes have led to the network of synteny revealed between tomato and
Arabidopsis
and predict that such networks of synteny will be common when making comparisons among higher plant taxa (e.g., families).
Neuroinvasion by a Creutzfeldt–Jakob disease agent in the absence of B cells and follicular dendritic cells
Authors: Shlomchik Mark J. , Radebold Klaus , Duclos Nicole , Manuelidis Laura
Published: July 2002

With the potential spread of bovine spongiform encephalopathy to people as a variant Creutzfeldt–Jakob disease (CJD), it becomes critical to identify cells in the periphery that carry infection. Initial work with scrapie agents suggested that B cells were central vectors for neuroinvasion. Subsequent studies indicated that B cells played an indirect role by promoting the development of follicular dendritic cells (FDCs) that accumulate abnormal prion protein (PrP). The mechanism for the role of FDCs, however, has not been clear. To further dissect potential B cell functions that contribute to neuroinvasion, we inoculated a CJD agent into mutant mice that (
i
) lacked B cells, (
ii
) had B cells unable to secrete Ig, or (
iii
) could secrete only IgM. Remarkably, all these mice developed disease with practically indistinguishable incubation times. The demonstration that neither immune complexes nor B cells were required for neuroinvasion from the periphery mandates a reanalysis of the accepted view of the essential role of B cells and FDC in these infections. Moreover, immune complexes were not required for the accumulation of pathologic PrP on the surface of FDCs, suggesting that PrP can bind to FDCs autonomously or by means of another factor. Wild-type mice had incubation times ≈50 days less than all mutant mice at the same peripheral doses, indicating that an intact immune system may increase agent uptake and delivery, but this condition is not essential. Specifically, the evidence to date suggests that IgG may enhance pivotal agent interactions with migratory myeloid cells.
An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction
Authors: Reid W. , Sadowska M. , Denaro F. , Rao S. , Foulke J. , Hayes N. , Jones O. , Doodnauth D. , Davis H. , Sill A. , O'Driscoll P. , Huso D. , Fouts T. , Lewis G. , Hill M. , Kamin-Lewis R. , Wei C. , Ray P. , Gallo R. C. , Reitz M. , Bryant J.
Published: July 2002

We report, to our knowledge, the first HIV type 1 (HIV-1) transgenic (Tg) rat. Expression of the transgene, consisting of an HIV-1 provirus with a functional deletion of
gag
and
pol
, is regulated by the viral long terminal repeat. Spliced and unspliced viral transcripts were expressed in lymph nodes, thymus, liver, kidney, and spleen, suggesting that Tat and Rev are functional. Viral proteins were identified in spleen tissue sections by immunohistochemistry and gp120 was present in splenic macrophages, T and B cells, and in serum. Clinical signs included wasting, mild to severe skin lesions, opaque cataracts, neurological signs, and respiratory difficulty. Histopathology included a selective loss of splenocytes within the periarterial lymphoid sheath, increased apoptosis of endothelial cells and splenocytes, follicular hyperplasia of the spleen, lymphocyte depletion of mesenteric lymph nodes, interstitial pneumonia, psoriatic skin lesions, and neurological, cardiac, and renal pathologies. Immunologically, delayed-type hypersensitivity response to keyhole limpet hemocyanin was diminished. By contrast, Ab titers and proliferative response to recall antigen (keyhole limpet hemocyanin) were normal. The HIV-1 Tg rat thus has many similarities to humans infected with HIV-1 in expression of viral genes, immune-response alterations, and pathologies resulting from infection. The HIV-1 Tg rat may provide a valuable model for some of the pathogenic manifestations of chronic HIV-1 diseases and could be useful in testing therapeutic regimens targeted to stages of viral replication subsequent to proviral integration.
Elevated arginase I expression in rat aortic smooth muscle cells increases cell proliferation
Authors: Wei Liu Hua , Wu Guoyao , Morris Sidney M. , Ignarro Louis J.
Published: July 2002

Arginase, which exists as the isoforms arginase I and II, catalyzes
the hydrolysis of arginine to ornithine and urea. Ornithine is the
principal precursor for production of polyamines, which are required
for cell proliferation. Rat aortic smooth muscle cells (RASMC) contain
constitutive arginase I, and arginase inhibitors cause inhibition of
cell proliferation. The objective of this study was to determine
whether the elevated expression of arginase I in RASMC causes increased
cell proliferation. RASMC were stably transfected with either rat
arginase I cDNA or a β-galactosidase control expression plasmid.
Western blots and arginase enzymatic assays revealed high-level
expression of cytosolic arginase I in arginase I-transfected RASMC.
Moreover, this observation was associated with the increased production
of urea and polyamines and higher rates of RASMC proliferation. The two
selective inhibitors of arginase,
N
G
-hydroxy-
l
-arginine and
S
-(2-boronoethyl)-
l
-cysteine, inhibited
arginase and decreased the production of urea and polyamines in
arginase I-transfected RASMC, all of which were associated with the
inhibition of cell proliferation. This study demonstrates that elevated
arginase I expression increases RASMC proliferation by mechanisms
involving increased production of polyamines. These observations
suggest that arginase I plays a potentially important role in
controlling RASMC proliferation.
Sexual dimorphism among bovine embryos in their ability to make the transition to expanded blastocyst and in the expression of the signaling molecule IFN-τ
Authors: Larson Melissa A. , Kimura Koji , Kubisch H. Michael , Roberts R. Michael
Published: July 2002

IFN-τ is a secretory product of trophectoderm of cattle, sheep,
and their relatives and is expressed for a few days in early pregnancy
after the blastocyst first forms. It serves to alert the mother that
she is pregnant. A delayed or less than robust IFN-τ signal is a
likely cause of embryonic loss. Here we have determined whether
blastocyst production of IFN-τ, which is encoded by a cluster of
genes on chromosome 9, differs between the sexes in cattle, as assessed
by culture of
in vitro
-derived embryos on two different
media, one complex (tissue culture medium 199 supplemented with serum)
with coculture support, the other relatively simple (synthetic
oviductal fluid plus albumin). With both media, female blastocysts
produced approximately double the amount of IFN-τ as males,
regardless of such variables as oocyte batch, blastocyst quality,
hatching, and length of time in culture. However, in either tissue
culture medium 199, which contains 5.5 mM
d
-glucose, or in
synthetic oviductal fluid, in the presence but not in the absence of
added glucose, significantly fewer female than male embryos were able
to progress from the morula/early blastocyst stage to more advanced
stages of development. It is possible that the differences between male
and female embryos both in their production of IFN-τ and in their
ability to progress in development in glucose-rich media are
manifestations of phenomena that occur
in vivo
and
provide plasticity in embryo selection during early pregnancy.
Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host
Authors: Tooley Aaron J. , Cai Yuping A. , Glazer Alexander N.
Published: July 2002

The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium (
Synechocystis
sp. PCC6803) was reconstituted in
Escherichia coli
. Cyanobacterial genes encoding enzymes required for the conversion of heme to the natural chromophore 3
Z
-phycocyanobilin, namely, heme oxygenase 1 and 3
Z
-phycocyanobilin:ferredoxin oxidoreductase, were expressed from a plasmid under control of the hybrid
trp
-
lac
(
trc
) promoter. Genes for the apoprotein (C-phycocyanin α subunit;
cpcA
) and the heterodimeric lyase (
cpcE
and
cpcF
) that catalyzes chromophore attachment were expressed from the
trc
promoter on a second plasmid. Upon induction, recombinant
E. coli
used the cellular pool of heme to produce holo-CpcA with spectroscopic properties qualitatively and quantitatively similar to those of the same protein produced endogenously in cyanobacteria. About a third of the apo-CpcA was converted to holo-CpcA. No significant bilin addition took place in a similarly engineered
E. coli
strain that lacks
cpcE
and
cpcF
. This approach should permit incisive analysis of many remaining questions in phycobiliprotein biosynthesis. These studies also demonstrate the feasibility of generating constructs of these proteins
in situ
for use as fluorescent protein probes in living cells.
The γ-aminobutyric acid type A (GABA
A
) receptor-associated protein (GABARAP) promotes GABA
A
receptor clustering and modulates the channel kinetics
Authors: Chen Li , Wang Hongbing , Vicini Stefano , Olsen Richard W.
Published: July 2002

A microtubule-associated protein, γ-aminobutyric acid type A
(GABA
A
) receptor-associated protein (GABARAP), was
previously identified as binding to the intracellular domain of
GABA
A
receptors by using the yeast two-hybrid screen. In
the present work, immunofluorescent staining and green fluorescent
protein-tagged receptor subunits showed that GABARAP is associated with
and promotes the clustering of GABA
A
receptors in QT-6
quail fibroblasts. The tubulin-binding motif of GABARAP and the γ2
subunit of the receptor are required. Disruption of microtubules
prevents the clustering in a time-dependent manner. When green
fluorescent protein-tagged α1 or γ2 subunit coexpressed with β2,
γ2L, and GABARAP was used, recordings from visually identified cells
revealed that clustered GABA
A
receptor had an
EC
50
of about 20 μM, vs. 5.7 μM for the diffuse
receptor. Clustered receptors deactivated faster and desensitized
slower than the diffuse receptors, because of decrease in the apparent
affinity of GABA binding. Different properties for clustered receptors
relative to unclustered receptors in heterologous cells suggest that
homologous differences between extrasynaptic and synaptic clustered
receptors in neurons may be due to the organization of the postsynaptic
machinery.
In vivo
transposition of
Minos
, a
Drosophila
mobile element, in mammalian tissues
Authors: Zagoraiou Laskaro , Drabek Dubravka , Alexaki Sofia , Guy Jacky A. , Klinakis Apostolos G. , Langeveld An , Skavdis George , Mamalaki Clio , Grosveld Frank , Savakis Charalambos
Published: July 2002

Transposable elements have been used widely in the past 20 years
for gene transfer and insertional mutagenesis in
Drosophila
. Transposon-based technology for gene
manipulation and genomic analysis currently is being adopted for
vertebrates. We tested the ability of
Minos
, a DNA
transposon from
Drosophila hydei
, to transpose in mouse
tissues. Two transgenic mouse lines were crossed, one expressing
Minos
transposase in lymphocytes under the control of
the CD2 promoter/locus control region and another carrying a
nonautonomous
Minos
transposon. Only mice containing
both transgenes show excision of the transposon and transposition into
new chromosomal sites in thymus and spleen cells. In addition,
expression of
Minos
transposase in embryonic fibroblast
cell lines derived from a transposon-carrying transgenic mouse resulted
in excision of the transposon. These results are a first step toward a
reversible insertional mutagenesis system in the mouse, opening the way
to develop powerful technologies for functional genomic analysis in
mammals.
Germ cell selection in genetic mosaics in
Drosophila melanogaster
Authors: Extavour Cassandra , García-Bellido Antonio
Published: July 2002

Heritable mutations in the germ line lead to genetically
heterogeneous, or mosaic, gonads. Many of the genes used in germ-line
development also play roles in somatic development [Saffman, E.
E. & Lasko, P. (1999)
Cell. Mol. Life Sci.
55,
1141–1163]. Mutations in these genes may have cellular phenotypes
throughout germ-line development leading to their differential
elimination or survival, as has been observed in somatic cells
[Morata, G. & Ripoll, P. (1975)
Dev. Biol.
42,
211–221]. We investigate whether mutations in heterozygosis are
subject to pregametic selection in the germ line. We initiated clones
of wild-type homozygous cells at different stages of development in
gonads heterozygous for eight different recessive chromosome
deficiencies. Here we show that cell selection takes place in mosaic
germ-line populations. This phenomenon represents a level of selection
that precedes and conditions subsequent zygotic selection by affecting
the genes available in the gametic population.
Meiotic recombination frequencies are affected by nutritional states in
Saccharomyces
cerevisiae
Authors: Abdullah Mohamad F. F. , Borts Rhona H.
Published: July 2002

Meiotic recombination in the yeast
Saccharomyces
cerevisiae
is initiated by programmed double-strand breaks at
selected sites throughout the genome (hotspots). α-Hotspots
are binding sites for transcription factors. Double-strand breaks at
α-hotspots require binding of transcription factor but not high
levels of transcription
per se
. We show that modulating
the production of the transcription factor Gcn4p by deletion or
constitutive transcription alters the rate of gene conversion
and crossing-over at
HIS4
. In addition, we show that
alterations in the metabolic state of the cell change the frequency of
gene conversion at
HIS4
in a Gcn4p-dependent manner. We
suggest that recombination data obtained from experiments using amino
acid and other biosynthetic genes for gene disruptions and/or as
genetic markers should be treated cautiously. The demonstration that
Gcn4p affects transcription of more than 500 genes and that the
recombinationally “hottest” ORFs tend to be Gcn4p-regulated
suggest that the metabolic state of a cell, especially with respect to
nitrogen metabolism, is a determinant of recombination rates.
This observation suggests that the effects of metabolic state
may be global and may account for some as yet unexplained features of
recombination in higher organisms, such as the differences in map
length between the sexes.
Induction of photosensitivity in neonatal rat pineal gland
Authors: Tosini Gianluca , Doyle Susan , Geusz Mike , Menaker Michael
Published: July 2002

Pineal glands removed from neonatal rats at 5, 7, and 9 days of age
and explanted into short-term culture, synthesized melatonin when
stimulated with norepinephrine (NE); their melatonin synthesis could
not be suppressed with bright white light. Dispersed pineal cell
cultures or pineal explants prepared from 1-day-old neonates and held
in culture for 7 or 9 days also synthesized melatonin when stimulated
with NE, but in these cases melatonin synthesis was significantly
suppressed by light, demonstrating that the pineals had become
photosensitive while in culture. The development of photosensitivity in
culture could be partially or completely abolished by the continuous
presence of 1 or 10 μm of NE in the culture medium. The pineals of
all nonmammalian vertebrates are photoreceptive, whereas those of
mammals do not normally respond to light. We hypothesize that a
mechanism to suppress pineal photosensitivity by using NE released from
sympathetic nerve endings evolved early in the history of mammals.
Localizing proteins in the cell from their phylogenetic profiles
Authors: Marcotte Edward M. , Xenarios Ioannis , van der Bliek Alexander M. , Eisenberg David
Published: July 2002

We introduce a computational method for identifying subcellular
locations of proteins from the phylogenetic distribution of the
homologs of organellar proteins. This method is based on the
observation that proteins localized to a given organelle by experiments
tend to share a characteristic phylogenetic distribution of their
homologs—a phylogenetic profile. Therefore any other protein can be
localized by its phylogenetic profile. Application of this method to
mitochondrial proteins reveals that nucleus-encoded proteins previously
known to be destined for mitochondria fall into three groups:
prokaryote-derived, eukaryote-derived, and organism-specific (i.e.,
found only in the organism under study). Prokaryote-derived
mitochondrial proteins can be identified effectively by their
phylogenetic profiles. In the yeast
Saccharomyces
cerevisiae
, 361 nucleus-encoded mitochondrial proteins can be
identified at 50% accuracy with 58% coverage. From these values and
the proportion of conserved mitochondrial genes, it can be inferred
that ≈630 genes, or 10% of the nuclear genome, is devoted to
mitochondrial function. In the worm
Caenorhabditis elegans,
we estimate that
there are ≈660 nucleus-encoded mitochondrial genes, or 4% of its
genome, with ≈400 of these genes contributed from the prokaryotic
mitochondrial ancestor. The large fraction of organism-specific and
eukaryote-derived genes suggests that mitochondria perform specialized
roles absent from prokaryotic mitochondrial ancestors. We observe
measurably distinct phylogenetic profiles among proteins from different
subcellular compartments, allowing the general use of prokaryotic
genomes in learning features of eukaryotic proteins.
A voltage-gated ion channel expressed specifically in spermatozoa
Authors: Quill Timothy A. , Ren Dejian , Clapham David E. , Garbers David L.
Published: July 2002

Calcium ions play a primary role in the regulation of sperm cell behavior. We report finding a voltage-gated ion channel (CatSper2) that is expressed in male germ cells but not in other cells. The putative channel contains 6 transmembrane segments, making it more similar to the voltage-gated potassium channels, but the ion selectivity pore domain sequence resembles that of a Ca
v
channel. The mRNA is expressed during the meiotic or postmeiotic stages of spermatogenesis, and the protein is localized to the sperm flagellum, suggesting a role in the regulation of sperm motility. The mRNA for the channel is present in mouse, rat, and human sperm cells, and the gene is found on chromosome 2 E5–F1 in the mouse and 15q13 in the human. Recently, another voltage-gated channel (CatSper) that has features similar to the one reported here was discovered. It also is expressed within the flagellum and is required for normal fertility of mice. However, expression of CatSper2 alone or coexpression with CatSper in cultured cells, or attempts to coimmunoprecipitate the two proteins from germ cells failed to demonstrate that these two unique but similar α-like subunits form either a homo- or heterotetramer. It is possible, therefore, that two independent α subunits, different from other known voltage-gated channels, regulate sperm motility.
Project normal: Defining normal variance in mouse gene expression
Authors: Pritchard Colin C. , Hsu Li , Delrow Jeffrey , Nelson Peter S.
Published: July 2002

The mouse has become an indispensable and versatile model organism for the study of development, genetics, behavior, and disease. The application of comprehensive gene expression profiling technologies to compare normal and diseased tissues or to assess molecular alterations resulting from various experimental interventions has the potential to provide highly detailed qualitative and quantitative descriptions of these processes. Ideally, to interpret experimental data, the magnitude and diversity of gene expression for the system under study should be well characterized, yet little is known about the normal variation of mouse gene expressionin vivo. To assess natural differences in murine gene expression, we used a 5406-clone spotted cDNA microarray to quantitate transcript levels in the kidney, liver, and testis from each of 6 normal male C57BL6 mice. We used ANOVA to compare the variance across the six mice to the variance among four replicate experiments performed for each mouse tissue. For the 6 kidney samples, 102 of 3,088 genes (3.3%) exhibited a statistically significant mouse variance at a level of 0.05. In the testis, 62 of 3,252 genes (1.9%) showed statistically significant variance, and in the liver, there were 21 of 2,514 (0.8%) genes with significantly variable expression. Immune-modulated, stress-induced, and hormonally regulated genes were highly represented among the transcripts that were most variable. The expression levels of several genes varied significantly in more than one tissue. These studies help to define the baseline level of variability in mouse gene expression and emphasize the importance of replicate microarray experiments.
Bacillus subtilis
arsenate reductase is structurally and functionally similar to low molecular weight protein tyrosine phosphatases
Authors: Bennett Matthew S. , Guan Zhi , Laurberg Martin , Su Xiao-Dong
Published: July 2002

Arsenate is an abundant oxyanion that, because of its ability to mimic the phosphate group, is toxic to cells. Arsenate reductase (EC
1.97.1.5
; encoded by the
arsC
gene in bacteria) participates to achieve arsenate resistance in both prokaryotes and yeast by reducing arsenate to arsenite; the arsenite is then exported by a specific transporter. The crystal structure of
Bacillus subtilis
arsenate reductase in the reduced form with a bound sulfate ion in its active site is solved at 1.6-Å resolution. Significant structural similarity is seen between arsenate reductase and bovine low molecular weight protein tyrosine phosphatase, despite very low sequence identity. The similarity is especially high between their active sites. It is further confirmed that this structural homology is relevant functionally by showing the phosphatase activity of the arsenate reductase
in vitro
. Thus, we can understand the arsenate reduction in the light of low molecular weight protein tyrosine phosphatase mechanism and also explain the catalytic roles of essential residues such as Cys-10, Cys-82, Cys-89, Arg-16, and Asp-105. A “triple cysteine redox relay” is proposed for the arsenate reduction mechanism.
Conservation of methylation reprogramming in mammalian development: Aberrant reprogramming in cloned embryos
Authors: Dean Wendy , Santos Fátima , Stojkovic Miodrag , Zakhartchenko Valeri , Walter Jörn , Wolf Eckhard , Reik Wolf
Published: July 2002

Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by
de novo
methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead,
de novo
methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.
A new screen for protein interactions reveals that the
Saccharomyces cerevisiae
high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter
Authors: Laser Heike , Bongards Christine , Schüller Jutta , Heck Stephanie , Johnsson Nils , Lehming Norbert
Published: July 2002

The split-ubiquitin assay detects protein interactions
in vivo
. To identify proteins interacting with Gal4p and
Tup1p, two transcriptional regulators, we converted the split-ubiquitin
assay into a generally applicable screen for binding partners of
specific proteins
in vivo
. A library of genomic
Saccharomyces cerevisiae
DNA fragments fused to the
N-terminal half of ubiquitin was constructed and transformed into yeast
strains carrying either Gal4p or Tup1p as a bait. Both proteins were
C-terminally extended by the C-terminal half of ubiquitin followed by a
modified Ura3p with an arginine in position 1, a destabilizing residue
in the N-end rule pathway. The bait fusion protein alone is stable and
enzymatically active. However, upon interaction with its prey, a
native-like ubiquitin is reconstituted. RUra3p is then cleaved off by
the ubiquitin-specific proteases and rapidly degraded by the N-end rule
pathway. In both screens, Nhp6B was identified as a protein in close
proximity to Gal4p as well as to Tup1p. Direct interaction between
either protein and Nhp6B was confirmed by coprecipitation assays.
Genetic analysis revealed that Nhp6B, a member of the HMG1 family of
DNA-binding proteins, can influence transcriptional activation as well
as repression at a specific locus in the chromosome of the yeast
S. cerevisiae
.
Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN
Authors: Yorita Kazuko , Matsuoka Takeshi , Misaki Hideo , Massey Vincent
Published: July 2002

Two arginine residues, Arg-181 and Arg-268, are conserved
throughout the known family of FMN-containing enzymes that catalyze the
oxidation of α-hydroxyacids. In the lactate oxidase from
Aerococcus viridans
, these residues have been changed to
lysine in two single mutations and in a double mutant form. In
addition, Arg-181 has been replaced by methionine to determine the
effect of removing the positive charge on the residue. The effects of
these replacements on the kinetic and thermodynamic properties are
reported. With all mutant forms, there are only small effects on the
reactivity of the reduced flavin with oxygen. On the other hand, the
efficiency of reduction of the oxidized flavin by
l
-lactate
is greatly reduced, particularly with the R268K mutant forms. The
results demonstrate the importance of the two arginine residues in the
binding of substrate and its interaction with the flavin, and are
consistent with a previous hypothesis that they also play a role of
charge neutralization in the transition state of substrate
dehydrogenation. The replacement of Arg-268 by lysine also results in a
slow conversion of the 8-CH
3
- substituent of FMN to yield
8-formyl-FMN, still tightly bound to the enzyme, and with significantly
different physical and chemical properties from those of the
FMN-enzyme.
Mutational studies on HslU and its docking mode with HslV
Authors: Song Hyun Kyu , Hartmann Claudia , Ramachandran Ravishankar , Bochtler Matthias , Behrendt Raymond , Moroder Luis , Huber Robert
Published: July 2002

HslVU is an ATP-dependent prokaryotic protease complex. Despite
detailed crystal and molecular structure determinations of free HslV
and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis
remained unclear, mainly because the productive complex of HslV and
HslU could not be unambiguously identified from the crystal data. In
the crystalline complex, the I domains of HslU interact with HslV.
Observations based on electron microscopy data were interpreted in the
light of the crystal structure to indicate an alternative mode of
association with the intermediate domains away from HslV. By generation
and analysis of two dozen HslU mutants, we find that the amidolytic and
caseinolytic activities of HslVU are quite robust to mutations on both
alternative docking surfaces on HslU. In contrast, HslVU activity
against the maltose-binding protein-SulA fusion protein depends on the
presence of the I domain and is also sensitive to mutations in the
N-terminal and C-terminal domains of HslU. Mutational studies around
the hexameric pore of HslU seem to show that it is involved in the
recognition/translocation of maltose-binding protein-SulA but not of
chromogenic small substrates and casein. ATP-binding site mutations,
among other things, confirm the essential role of the “sensor
arginine” (R393) and the “arginine finger” (R325) in the
ATPase action of HslU and demonstrate an important role for E321.
Additionally, we report a better refined structure of the HslVU complex
crystallized along with resorufin-labeled casein.
Mechanisms underlying rapid experience-dependent plasticity in the human visual cortex
Authors: Boroojerdi Babak , Battaglia Fortunato , Muellbacher Wolf , Cohen Leonardo G.
Published: July 2002

Visual deprivation induces a rapid increase in visual cortex
excitability that may result in better consolidation of spatial memory
in animals and in lower visual recognition thresholds in humans.
γ-Aminobutyric acid (GABA)ergic,
N
-methyl-
d
-aspartate (NMDA), and cholinergic
receptors are thought to be involved in visual cortex plasticity in
animal studies. Here, we used a pharmacological approach and found that
lorazepam (which enhances GABA
A
receptor function by acting
as a positive allosteric modulator), dextrometorphan (NMDA receptor
antagonist), and scopolamine (muscarinic receptor antagonist) blocked
rapid plastic changes associated with light deprivation. These findings
suggest the involvement of GABA, NMDA, and cholinergic receptors in
rapid experience-dependent plasticity in the human visual cortex.
Cell injury releases endogenous adjuvants that stimulate cytotoxic T cell responses
Authors: Shi Yan , Zheng Wanyong , Rock Kenneth L.
Published: July 2002

General immunostimulants (adjuvants) are essential for generating immunity to many antigens. In bacterial infections, adjuvants are provided by components of the microorganism, e.g., lipopolysaccharide. However, it is unclear what provides the adjuvant effect for immune responses that are generated to tumors and many viruses. Here we show that cell injury and death of tumor or even normal cells provide a potent adjuvant effect for the stimulation of cytotoxic T lymphocyte responses. This adjuvant activity is constitutively present in the cytoplasm of cells and is increased in the cytoplasm of cells dying by apoptosis. The release of these components stimulates immune responses both locally and at a distance, and provides a simple mechanism to alert the immune system to potential danger in almost all pathological situations.
Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis.
Authors: Ito K , Ebihara K , Uno M , Nakamura Y
Published: July 2002

Translation termination requires two codon-specific polypeptide release factors in prokaryotes and one omnipotent factor in eukaryotes. Sequences of 17 different polypeptide release factors from prokaryotes and eukaryotes were compared. The prokaryotic release factors share residues split into seven motifs. Conservation of many discrete, perhaps critical, amino acids is observed in eukaryotic release factors, as well as in the C-terminal portion of elongation factor (EF) G. Given that the C-terminal domains of EF-G interacts with ribosomes by mimicry of a tRNA structure, the pattern of conservation of residues in release factors may reflect requirements for a tRNA-mimicry for binding to the A site of the ribosome. This mimicry would explain why release factors recognize stop codons and suggests that all prokaryotic and eukaryotic release factors evolved from the progenitor of EF-G.
Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element.
Authors: Lu F M , Lux S E
Published: July 2002

Notch is a transmembrane receptor that plays a critical role in cell fate determination. In Drosophila, Notch binds to and signals through Suppressor of Hairless. A mammalian homologue of Suppressor of Hairless, named CBF1 (or RBPJk), is a ubiquitous transcription factor whose function in mammalian Notch signaling is unknown. To determine whether mammalian Notch can stimulate transcription through a CBF1-responsive element (RE), we cotransfected a CBF1-RE-containing chloramphenicol acetyltransferase reporter and N1(deltaEC), a constitutively active form of human Notch1 lacking the extracellular domain, into DG75, COS-1, HeLa, and 293T cells, which all contain endogenous CBF1. N1(deltaEC) dramatically increased chloramphenicol acetyltransferase activity in these cells, indicating functional coupling of Notch1 and CBF1. The activity was comparable to that produced by the Epstein-Barr virus protein EBNA2, a well-characterized, potent transactivator of CBF1. To test whether CBF1 and Notch1 interact physically, we tagged CBF1 with an epitope from the influenza virus hemagglutinin or with the N-terminal domain of gal4, and transfected the tagged CBF1 plus N1(deltaEC) into COS-1 cells. Cell lysates were immunoprecipitated and immunoblotted with several anti-Notch1 antibodies [to detect N1(deltaEC)] or with antibodies to hemagglutinin or gal4 (to detect CBF1). Each immunoprecipitate contained a complex of N1(deltaEC) and CBF1. In summary, we find that the truncated, active form of human Notch1, N1(deltaEC), binds CBF1 and activates transcription through a CBF1-RE-containing promoter. We conclude that CBF1 is a critical downstream protein in the human Notch1 signaling pathway.
A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide.
Authors: Yim M B , Kang J H , Yim H S , Kwak H S , Chock P B , Stadtman E R
Published: July 2002

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Gly-93--> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the FALS mutant, G93A, and wild-type cDNA of human Cu,Zn-SOD, overexpressed them in Sf9 insect cells, purified the proteins, and studied their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as substrate. Our results showed that both enzymes contain one copper ion per subunit and have identical dismutation activity. However, the free radical-generating function of the G93A mutant, as measured by the spin trapping method, is enhanced relative to that of the wild-type enzyme, particularly at lower H2O2 concentrations. This is due to a small, but reproducible, decrease in the value of Km for H2O2 for the G93A mutant, while the kcat is identical for both enzymes. Thus, the ALS symptoms observed in G93A transgenic mice are not caused by the reduction of Cu,Zn-SOD activity with the mutant enzyme; rather, it is induced by a gain-of-function, an enhancement of the free radical-generating function. This is consistent with the x-ray crystallographic studies showing the active channel of the FALS mutant is slightly larger than that of the wild-type enzyme; thus, it is more accessible to H2O2. This gain-of-function, in part, may provide an explanation for the association between ALS and Cu,Zn-SOD mutants.
A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos.
Authors: Ault K T , Dirksen M L , Jamrich M
Published: July 2002

The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.