bluuebunny/crossref_metadata_2025 huggingface dataset:
Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse
Authors: Liu Wen Sheng , Pesold Christine , Rodriguez Miguel A. , Carboni Giovanni , Auta James , Lacor Pascal , Larson John , Condie Brian G. , Guidotti Alessandro , Costa Erminio
Published: July 2002

Heterozygous reeler mice (HRM) haploinsufficient for reelin express
≈50% of the brain reelin content of wild-type mice, but are
phenotypically different from both wild-type mice and homozygous reeler
mice. They exhibit, (
i
) a down-regulation of glutamic
acid decarboxylase 67 (GAD
67
)-positive neurons in some but
not every cortical layer of frontoparietal cortex (FPC),
(
ii
) an increase of neuronal packing density and a
decrease of cortical thickness because of neuropil hypoplasia,
(
iii
) a decrease of dendritic spine expression density
on basal and apical dendritic branches of motor FPC layer III pyramidal
neurons, and (
iv
) a similar decrease in dendritic spines
expressed on the basal dendrite branches of CA1 pyramidal neurons of
the hippocampus. To establish whether the defect of GAD
67
down-regulation observed in HRM is responsible for neuropil hypoplasia
and decreased dendritic spine density, we studied heterozygous
GAD
67
knockout mice (HG
67
M). These mice
exhibited a down-regulation of GAD
67
mRNA expression in FPC
(about 50%), but they expressed normal amounts of reelin and had no
neuropil hypoplasia or down-regulation of dendritic spine expression.
These findings, coupled with electron-microscopic observations that
reelin colocalizes with integrin receptors on dendritic spines, suggest
that reelin may be a factor in the dynamic expression of cortical
dendritic spines perhaps by promoting integrin receptor clustering.
These findings are interesting because the brain neurochemical and
neuroanatomical phenotypic traits exhibited by the HRM are in several
ways similar to those found in postmortem brains of psychotic patients.
Neural mechanisms of planning: A computational analysis using event-related fMRI
Authors: Fincham Jon M. , Carter Cameron S. , van Veen Vincent , Stenger V. Andrew , Anderson John R.
Published: July 2002

To investigate the neural mechanisms of planning, we used a novel adaptation of the Tower of Hanoi (TOH) task and event-related functional MRI. Participants were trained in applying a specific strategy to an isomorph of the five-disk TOH task. After training, participants solved novel problems during event-related functional MRI. A computational cognitive model of the task was used to generate a reference time series representing the expected blood oxygen level-dependent response in brain areas involved in the manipulation and planning of goals. This time series was used as one term within a general linear modeling framework to identify brain areas in which the time course of activity varied as a function of goal-processing events. Two distinct time courses of activation were identified, one in which activation varied parametrically with goal-processing operations, and the other in which activation became pronounced only during goal-processing intensive trials. Regions showing the parametric relationship comprised a frontoparietal system and include right dorsolateral prefrontal cortex [Brodmann's area (BA 9)], bilateral parietal (BA 40/7), and bilateral premotor (BA 6) areas. Regions preferentially engaged only during goal-intensive processing include left inferior frontal gyrus (BA 44). The implications of these results for the current model, as well as for our understanding of the neural mechanisms of planning and functional specialization of the prefrontal cortex, are discussed.
VIPP1
, a nuclear gene of
Arabidopsis thaliana
essential for thylakoid membrane formation
Authors: Kroll Daniela , Meierhoff Karin , Bechtold Nicole , Kinoshita Mikio , Westphal Sabine , Vothknecht Ute C. , Soll Jürgen , Westhoff Peter
Published: July 2002

The conversion of light to chemical energy by the process of
photosynthesis is localized to the thylakoid membrane network in plant
chloroplasts. Although several pathways have been described that target
proteins into and across the thylakoids, little is known about the
origin of this membrane system or how the lipid backbone of the
thylakoids is transported and fused with the target membrane. Thylakoid
biogenesis and maintenance seem to involve the flow of membrane
elements via vesicular transport. Here we show by mutational analysis
that deletion of a single gene called
VIPP1
(vesicle-inducing protein in plastids 1) is deleterious to thylakoid
membrane formation. Although VIPP1 is a hydrophilic protein it is found
in both the inner envelope and the thylakoid membranes. In
VIPP1
deletion mutants vesicle formation is abolished.
We propose that
VIPP1
is essential for the maintenance
of thylakoids by a transport pathway not previously recognized.
The Sm domain is an ancient RNA-binding motif with oligo(U) specificity
Authors: Achsel Tilmann , Stark Holger , Lührmann Reinhard
Published: July 2002

Sm and Sm-like proteins are members of a family of small proteins
that is widespread throughout eukaryotic kingdoms. These proteins form
heteromers with one another and bind, as heteromeric complexes, to
various RNAs, recognizing primarily short U-rich stretches.
Interestingly, completion of several genome projects revealed that
archaea also contain genes that may encode Sm-like proteins. Herein, we
studied the properties of one Sm-like protein derived from the
archaebacterium
Archaeoglobus fulgidus
and overexpressed
in
Escherichia coli
. This single small protein closely
reflects the properties of an Sm or Sm-like protein heteromer. It binds
to RNA with a high specificity for oligo(U), and assembles onto the RNA
to form a complex that exhibits, as judged by electron microscopy, a
ring-like structure similar to the ones observed with the Sm core
ribonucleoprotein and the like Sm (LSm) protein heteromer. Importantly,
multivariate statistical analysis of negative-stain
electron-microscopic images revealed a sevenfold symmetry for the
observed ring structure, indicating that the proteins form a
homoheptamer. These results support the structural model of the Sm
proteins derived from crystallographic studies on Sm heterodimers and
demonstrate that the Sm protein family evolved from a single ancestor
that was present before the eukaryotic and archaeal kingdoms separated.
Role of poly(ADP-ribosyl)ation in DNA-PKcs- independent V(D)J recombination
Authors: Brown Matthew L. , Franco Daniel , Bürkle Alexander , Chang Yung
Published: July 2002

V(D)J recombination is critical to the generation of a functional immune system. Intrinsic to the assembly of antigen receptor genes is the formation of endogenous DNA double-strand breaks, which normally are excluded from the cellular surveillance machinery because of their sequestration in a synaptic complex and/or rapid resolution. In cells deficient in double-strand break repair, such recombination-induced breaks fail to be joined promptly and therefore are at risk of being recognized as DNA damage. Poly(ADP-ribose) polymerase-1 is an important factor in the maintenance of genomic integrity and is believed to play a central role in DNA repair. Here we provide visual evidence that in a recombination inducible severe combined immunodeficient cell line poly(ADP-ribose) formation occurs during the resolution stage of V(D)J recombination where nascent opened coding ends are generated. Poly(ADP-ribose) formation appears to facilitate coding end resolution. Furthermore, formation of Mre11 foci coincide with these areas of poly(ADP-ribosyl)ation. In contrast, such a response is not observed in wild-type cells possessing a functional catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Thus, V(D)J recombination invokes a DNA damage response in cells lacking DNA-PKcsactivity, which in turn promotes DNA-PKcs-independent resolution of recombination intermediates.
Cell membrane GM
1
ganglioside is a functional coreceptor for fibroblast growth factor 2
Authors: Rusnati Marco , Urbinati Chiara , Tanghetti Elena , Dell'Era Patrizia , Lortat-Jacob Hugues , Presta Marco
Published: July 2002

Free gangliosides bind fibroblast growth factor 2 (FGF2), thus preventing cell interaction and biological activity of the growth factor in endothelial cells. Here we investigated the role of cell-associated gangliosides in mediating the biological activity of FGF2. Treatment of endothelial cells of different origin with the ganglioside biosynthesis inhibitors fumonisin B
1
, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or D-1-threo-1-phenyl-2-hexa-decanoylamino-3-pyrrolidino-1-propanol-HCl, impairs their capacity to proliferate when exposed to FGF2. Also, the mitogenic activity of FGF2 is inhibited by the GM
1
-binding cholera toxin B subunit (CTB). Conversely, overloading of endothelial GM 7373 cell membranes with exogenous GM
1
causes a 10-fold increase of the mitogenic potency of FGF2.
125
I-FGF2 binds to cell membrane GM
1
(
K
d
= 3 nM) in complex ganglioside/heparan sulfate-deficient Chinese hamster ovary (CHO)-K1-pgsA745 cell mutants that were overloaded with exogenous GM
1
. Moreover, FGF2 competes with FITC-CTB for the binding to cell membrane GM
1
in different CHO cell lines independently of their capacity to express heparan sulfate proteoglycans. Conversely, CTB inhibits cell proliferation triggered by FGF2 in CHO cells overexpressing the tyrosine kinase FGF receptor 1. Finally, GM
1
-overloading confers to FGF receptor 1-transfected, complex ganglioside-deficient CHO-K1 cell mutants the capacity to proliferate when stimulated by FGF2. This proliferation is inhibited by CTB. Cell proliferation triggered by serum or by phorbol 12-myristate 13-acetate is instead independent of the cell membrane ganglioside milieu. In conclusion, cell membrane GM
1
binds FGF2 and is required for the mitogenic activity of the growth factor. Our data indicate that cell-associated gangliosides may act as functional FGF2 co-receptors in different cell types.
The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis
Authors: Bellizzi John J. , Widom Joanne , Kemp Christopher , Lu Jui-Yun , Das Amit K. , Hofmann Sandra L. , Clardy Jon
Published: July 2002

Mutations in palmitoyl-protein thioesterase 1 (PPT1), a lysosomal enzyme that removes fatty acyl groups from cysteine residues in modified proteins, cause the fatal inherited neurodegenerative disorder infantile neuronal ceroid lipofuscinosis. The accumulation of undigested substrates leads to the formation of neuronal storage bodies that are associated with the clinical symptoms. Less severe forms of PPT1 deficiency have been found recently that are caused by a distinct set of PPT1 mutations, some of which retain a small amount of thioesterase activity. We have determined the crystal structure of PPT1 with and without bound palmitate by using multiwavelength anomalous diffraction phasing. The structure reveals an α/β-hydrolase fold with a catalytic triad composed of Ser115-His289-Asp233 and provides insights into the structural basis for the phenotypes associated with PPT1 mutations.
Skp2 is oncogenic and overexpressed in human cancers
Authors: Gstaiger Matthias , Jordan Richard , Lim Megan , Catzavelos Charles , Mestan Jürgen , Slingerland Joyce , Krek Wilhelm
Published: July 2002

Skp2 is a member of the F-box family of substrate-recognition
subunits of SCF ubiquitin–protein ligase complexes that has been
implicated in the ubiquitin-mediated degradation of several key
regulators of mammalian G
1
progression, including the
cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor
suppressor protein. In this study, we examined Skp2 and p27 protein
expression by immunohistochemistry in normal oral epithelium and in
different stages of malignant oral cancer progression, including
dysplasia and oral squamous cell carcinoma. We found that increased
levels of Skp2 protein are associated with reduced p27 in a subset of
oral epithelial dysplasias and carcinomas compared with normal
epithelial controls. Tumors with high Skp2 (>20% positive cells)
expression invariably showed reduced or absent p27 and tumors with high
p27 (>20% positive cells) expression rarely showed Skp2 positivity.
Increased Skp2 protein levels were not always correlated with increased
cell proliferation (assayed by Ki-67 staining), suggesting that
alterations of Skp2 may contribute to the malignant phenotype without
affecting proliferation. Skp2 protein overexpression may lead to
accelerated p27 proteolysis and contribute to malignant progression
from dysplasia to oral epithelial carcinoma. Moreover, we also
demonstrate that Skp2 has oncogenic potential by showing that Skp2
cooperates with H-Ras
G12V
to malignantly transform primary
rodent fibroblasts as scored by colony formation in soft agar and tumor
formation in nude mice. The observations that Skp2 can mediate
transformation and is up-regulated during oral epithelial
carcinogenesis support a role for Skp2 as a protooncogene in human
tumors.
A monomeric red fluorescent protein
Authors: Campbell Robert E. , Tour Oded , Palmer Amy E. , Steinbach Paul A. , Baird Geoffrey S. , Zacharias David A. , Tsien Roger Y.
Published: July 2002

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of
Aequorea
green fluorescent protein (GFP) to dimerize, the obligate dimerization of
Renilla
GFP, and the obligate tetramerization of the red fluorescent protein from
Discosoma
(DsRed). Although the weak dimerization of
Aequorea
GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.
Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet β cells
Authors: Patterson George H. , Knobel Susan M. , Arkhammar Per , Thastrup Ole , Piston David W.
Published: July 2002

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be ≈30 μM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be ≈7 μM. Likewise, the NAD(P)H increase of the mitochondrial domain is ≈60 μM and is delayed with respect to the change in cytoplasmic NAD(P)H by ≈20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.
Nuclear structure in normal and Bloom syndrome cells
Authors: Yankiwski Victor , Marciniak Robert A. , Guarente Leonard , Neff Norma F.
Published: July 2002

Bloom syndrome (BS) is a rare cancer-predisposing disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. BLM, the protein altered in BS, is a RecQ DNA helicase. This report shows that BLM is found in the nucleus of normal human cells in the nuclear domain 10 or promyelocytic leukemia nuclear bodies. These structures are punctate depots of proteins disrupted upon viral infection and in certain human malignancies. BLM is found primarily in nuclear domain 10 except during S phase when it colocalizes with the Werner syndrome gene product, WRN, in the nucleolus. BLM colocalizes with a select subset of telomeres in normal cells and with large telomeric clusters seen in simian virus 40-transformed normal fibroblasts. During S phase, BS cells expel micronuclei containing sites of DNA synthesis. BLM is likely to be part of a DNA surveillance mechanism operating during S phase.
Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells
Authors: Rocha Gerson M. , Michea Luis F. , Peters Eugenia M. , Kirby Martha , Xu Yuhui , Ferguson Douglas R. , Burg Maurice B.
Published: July 2002

Antipyretic analgesics, taken in large doses over a prolonged
period, cause a specific form of kidney disease, characterized by
papillary necrosis and interstitial scarring. Epidemiological evidence
incriminated mixtures of drugs including aspirin (ASA), phenacetin, and
caffeine. The mechanism of toxicity is unclear. We tested the effects
of ASA, acetaminophen (APAF, the active metabolite of phenacetin),
caffeine, and other related drugs individually and in combination on
mouse inner medullary collecting duct cells (mIMCD3). The number of
rapidly proliferating cells was reduced by ≈50% by 0.5 mM ASA,
salicylic acid, or APAF. The drugs had less effect on confluent cells,
which proliferate slowly. Thus, the slow
in vivo
turnover of IMCD cells could explain why clinical toxicity requires
very high doses of these drugs over a very long period. Caffeine
greatly potentiated the effect of acetaminophen, pointing to a
potential danger of the mixture. Cyclooxygenase (COX) inhibitors,
indomethacin and NS-398, did not reduce cell number except at
concentrations greatly in excess of those that inhibit COX. Therefore,
COX inhibition alone is not toxic. APAF arrests most cells in late
G
1
and S and produces a mixed form of cell death with both
oncosis (swollen cells and nuclei) and apoptosis. APAF is known
to inhibit the synthesis of DNA and cause chromosomal aberrations due
to inhibition of ribonucleotide reductase. Such effects of APAF might
account for renal medullary cell death
in vivo
and
development of uroepithelial tumors from surviving cells that have
chromosomal aberrations.
Using electroactive substrates to pattern the attachment of two different cell populations
Authors: Yousaf Muhammad N. , Houseman Benjamin T. , Mrksich Milan
Published: July 2002

This report describes the development of an electroactive mask that
permits the patterning of two different cell populations to a single
substrate. This mask is based on a self-assembled monolayer of
alkanethiolates on gold that could be switched from a state that
prevents the attachment of cells to a state that promotes the
integrin-mediated attachment of cells. Monolayers were patterned into
regions having this electroactive monolayer and a second set of regions
that were adhesive. After Swiss 3T3 fibroblasts had attached to the
adhesive regions of this substrate, the second set of regions was
activated electrically to permit the attachment of a second population
of fibroblast cells. This method provides a general strategy for
patterning the attachment of multiple cell types and will be important
for studying heterotypic cell-cell interactions.
Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca
2+
release channels
Authors: Yang Jun , McBride Sean , Mak Don-On Daniel , Vardi Noga , Palczewski Krzysztof , Haeseleer Françoise , Foskett J. Kevin
Published: July 2002

The inositol trisphosphate (Ins
P

3

) receptor (Ins
P
3
R) is a ubiquitously expressed intracellular Ca
2+
channel that mediates complex cytoplasmic Ca
2+
signals, regulating diverse cellular processes, including synaptic plasticity. Activation of the Ins
P
3
R channel is normally thought to require binding of Ins
P
3
derived from receptor-mediated activation of phosphatidylinositol lipid hydrolysis. Here we identify a family of neuronal Ca
2+
-binding proteins as high-affinity protein agonists of the Ins
P
3
R, which bind to the channel and activate gating in the absence of Ins
P
3
. CaBP/caldendrin, a subfamily of the EF-hand-containing neuronal calcium sensor family of calmodulin-related proteins, bind specifically to the Ins
P
3
-binding region of all three Ins
P
3
R channel isoforms with high affinity (K
a
≈ 25 nM) in a Ca
2+
-dependent manner (K
a
≈ 1 μM). Binding activates single-channel gating as efficaciously as Ins
P
3
, dependent on functional EF-hands in CaBP. In contrast, calmodulin neither bound with high affinity nor activated channel gating. CaBP1 and the type 1 Ins
P
3
R associate in rat whole brain and cerebellum lysates, and colocalize extensively in subcellular regions in cerebellar Purkinje neurons. Thus, Ins
P
3
R-mediated Ca
2+
signaling in cells is possible even in the absence of Ins
P
3
generation, a process that may be particularly important in responding to and shaping changes in intracellular Ca
2+
concentration by Ins
P
3
-independent pathways and for localizing Ins
P
3
-mediated Ca
2+
signals to individual synapses.
TheiscSgene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H
Authors: Mihara Hisaaki , Kato Shin-ichiro , Lacourciere Gerard M. , Stadtman Thressa C. , Kennedy Robert A. J. D. , Kurihara Tatsuo , Tokumoto Umechiyo , Takahashi Yasuhiro , Esaki Nobuyoshi
Published: July 2002

Three NifS-like proteins, IscS, CSD, and CsdB, fromEscherichia colicatalyze the removal of sulfur and selenium froml-cysteine andl-selenocysteine, respectively, to forml-alanine. These enzymes are proposed to function as sulfur-delivery proteins for iron-sulfur cluster, thiamin, 4-thiouridine, biotin, and molybdopterin. Recently, it was reported that selenium mobilized from free selenocysteine is incorporated specifically into a selenoprotein and tRNAin vivo, supporting the involvement of the NifS-like proteins in selenium metabolism. We here report evidence that a strain lacking IscS is incapable of synthesizing 5-methylaminomethyl-2-selenouridine and its precursor 5-methylaminomethyl-2-thiouridine (mnm5s2U) in tRNA, suggesting that the sulfur atom released froml-cysteine by the action of IscS is incorporated into mnm5s2U. In contrast, neither CSD nor CsdB was essential for production of mnm5s2U and 5-methylaminomethyl-2-selenouridine. The lack of IscS also caused a significant loss of the selenium-containing polypeptide of formate dehydrogenase H. Together, these results suggest a dual function of IscS in sulfur and selenium metabolism.
Operons in
Escherichia coli
: Genomic analyses and predictions
Authors: Salgado Heladia , Moreno-Hagelsieb Gabriel , Smith Temple F. , Collado-Vides Julio
Published: July 2002

The rich knowledge of operon organization in
Escherichia coli
, together with the completed chromosomal sequence of this bacterium, enabled us to perform an analysis of distances between genes and of functional relationships of adjacent genes in the same operon, as opposed to adjacent genes in different transcription units. We measured and demonstrated the expected tendencies of genes within operons to have much shorter intergenic distances than genes at the borders of transcription units. A clear peak at short distances between genes in the same operon contrasts with a flat frequency distribution of genes at the borders of transcription units. Also, genes in the same operon tend to have the same physiological functional class. The results of these analyses were used to implement a method to predict the genomic organization of genes into transcription units. The method has a maximum accuracy of 88% correct identification of pairs of adjacent genes to be in an operon, or at the borders of transcription units, and correctly identifies around 75% of the known transcription units when used to predict the transcription unit organization of the
E. coli
genome. Based on the frequency distance distributions, we estimated a total of 630 to 700 operons in
E. coli
. This step opens the possibility of predicting operon organization in other bacteria whose genome sequences have been finished.
Refractoriness to melatonin occurs independently at multiple brain sites in Siberian hamsters
Authors: Freeman David A. , Zucker Irving
Published: July 2002

The mid-winter development of refractoriness to melatonin
(Mel) triggers recrudescence of the atrophied reproductive apparatus of
rodents. As a consequence, over-wintering animals become reproductively
competent just before the onset of spring conditions favorable for
breeding. The neural target tissues that cease to respond to winter Mel
signals have not been identified. We now report that the
suprachiasmatic nucleus of the hypothalamus, which contains the
principal circadian clock, and the reuniens and paraventricular nuclei
of the thalamus, each independently becomes refractory to melatonin.
Small implants of Mel that were left in place for 40 wk and that act
locally on these brain nuclei, induced testicular regression within 6
wk in male Siberian hamsters; 12 wk later Mel implants no longer
suppressed reproduction and gonadal recrudescence ensued. Hamsters that
were then given a systemic Mel infusion s.c. immediately initiated a
second gonadal regression, implying that neurons at each site become
refractory to Mel without compromising responsiveness of other Mel
target tissues. Refractoriness occurs locally and independently at each
neural target tissue, rather than in a separate “refractoriness”
substrate. Restricted, target-specific actions of Mel are consistent
with the independent regulation by day length of the several behavioral
and physiological traits that vary seasonally in mammals.
Measuring absolute expression with microarrays with a calibrated reference sample and an extended signal intensity range
Authors: Dudley Aimée M. , Aach John , Steffen Martin A. , Church George M.
Published: July 2002

Gene expression ratios derived from spotted-glass microarray experiments have become invaluable to researchers by providing sensitive and comprehensive indicators of the molecular underpinnings of cell behaviors and states. However, several drawbacks to this form of data have been noted, including the inability to relate ratios to absolute expression levels or to compare experimental conditions not measured with the same control. In this study we demonstrate a method for overcoming these obstacles. First, instead of cohybridizing labeled experimental and control samples, we hybridize each sample against labeled oligos complementary to every microarray feature. Ratios between sample intensities and intensities of the oligo reference measure sample RNA levels on a scale that relates to their absolute abundance, instead of to the variable and unknown abundances of a cDNA reference. We demonstrate that results from this type of hybridization are accurate and retain absolute abundance information far better than conventional microarray ratios. Next, to ensure the accurate measurement of sample and oligo reference intensities, which may differ by several orders of magnitude, we use a linear regression algorithm, implemented in a freely available
perl
script, to combine the linear ranges of multiple scans taken at different scanner sensitivity settings onto an extended linear scale. We discuss future applications of our method to measure RNA expression on the absolute scale of number of transcripts per cell from any organism for which oligo-based spotted-glass microarrays are available.
Bacteriophage T4 gene 41 helicase and gene 59 helicase-loading protein: A versatile couple with roles in replication and recombination
Authors: Jones Charles E. , Mueser Timothy C. , Dudas Kathleen C. , Kreuzer Kenneth N. , Nossal Nancy G.
Published: July 2002

Bacteriophage T4 uses two modes of replication initiation: origin-dependent replication early in infection and recombination-dependent replication at later times. The same relatively simple complex of T4 replication proteins is responsible for both modes of DNA synthesis. Thus the mechanism for loading the T4 41 helicase must be versatile enough to allow it to be loaded on R loops created by transcription at several origins, on D loops created by recombination, and on stalled replication forks. T4 59 helicase-loading protein is a small, basic, almost completely α-helical protein whose N-terminal domain has structural similarity to high mobility group family proteins. In this paper we review recent evidence that 59 protein recognizes specific structures rather than specific sequences. It binds and loads the helicase on replication forks and on three- and four-stranded (Holliday junction) recombination structures, without sequence specificity. We summarize our experiments showing that purified T4 enzymes catalyze complete unidirectional replication of a plasmid containing the T4
ori(uvsY)
origin, with a preformed R loop at the position of the R loop identified at this origin
in vivo
. This replication depends on the 41 helicase and is strongly stimulated by 59 protein. Moreover, the helicase-loading protein helps to coordinate leading and lagging strand synthesis by blocking replication on the
ori(uvsY)
R loop plasmid until the helicase is loaded. The T4 enzymes also can replicate plasmids with R loops that do not have a T4 origin sequence, but only if the R loops are within an easily unwound DNA sequence.
Regulated transposition of a fish transposon in the mouse germ line
Authors: Fischer Sylvia E. J. , Wienholds Erno , Plasterk Ronald H. A.
Published: July 2002

Tc1/
mariner
elements are able to transpose in
species other than the host from which they were isolated. As potential
vectors for insertional mutagenesis and transgenesis of the mouse,
these cut-and-paste transposons were tested for their ability to
transpose in the mouse germ line. First, the levels of activity of
several Tc1/
mariner
elements in mammalian cells were
compared; the reconstructed fish transposon Sleeping Beauty (SB) was
found to be an order of magnitude more efficient than the other tested
transposons. SB then was introduced into the mouse germ line as a
two-component system: one transgene for the expression of the
transposase in the male germ line and a second transgene carrying a
modified transposon. In 20% of the progeny of double transgenic male
mice the transposon had jumped from the original chromosomal position
into another locus. Analysis of the integration sites shows that these
jumps indeed occurred through the action of SB transposase, and that SB
has a strong preference for intrachromosomal transposition. Analysis of
the excision sites suggests that double-strand breaks in haploid
spermatids are repaired via nonhomologous end joining. The SB system
may be a powerful tool for transposon mutagenesis of the mouse germ
line.
Genetic susceptibility to tuberculosis in Africans: A genome-wide scan
Authors: Bellamy Richard , Beyers Nulda , McAdam Keith P. W. J. , Ruwende Cyril , Gie Robert , Samaai Priscilla , Bester Danite , Meyer Mandy , Corrah Tumani , Collin Matthew , Camidge D. Ross , Wilkinson David , Hoal-van Helden Eileen , Whittle Hilton C. , Amos William , van Helden Paul , Hill Adrian V. S.
Published: July 2002

Human genetic variation is an important determinant of the outcome of infection with
Mycobacterium tuberculosis
. We have conducted a two-stage genome-wide linkage study to search for regions of the human genome containing tuberculosis-susceptibility genes. This approach uses sibpair families that contain two full siblings who have both been affected by clinical tuberculosis. For any chromosomal region containing a major tuberculosis-susceptibility gene, affected sibpairs inherit the same parental alleles more often than expected by chance. In the first round of the screen, 299 highly informative genetic markers, spanning the entire human genome, were typed in 92 sibpairs from The Gambia and South Africa. Seven chromosomal regions that showed provisional evidence of coinheritance with clinical tuberculosis were identified. To identify whether any of these regions contained a potential tuberculosis-susceptibility gene, 22 markers from these regions were genotyped in a second set of 81 sibpairs from the same countries. Markers on chromosomes 15q and Xq showed suggestive evidence of linkage (lod = 2.00 and 1.77, respectively) to tuberculosis. The potential identification of susceptibility loci on both chromosomes 15q and Xq was supported by an independent analysis designated common ancestry using microsatellite mapping. These results indicate that genome-wide linkage analysis can contribute to the mapping and identification of major genes for multifactorial infectious diseases of humans. An X chromosome susceptibility gene may contribute to the excess of males with tuberculosis observed in many different populations.
The type I BMP receptor
BmprIB
is essential for female reproductive function
Authors: Yi Soyun E. , LaPolt Philip S. , Yoon Byeong S. , Chen Jean Y.-C. , Lu John K. H. , Lyons Karen M.
Published: July 2002

Maintenance of female reproductive competence depends on the actions of several hormones and signaling factors. Recent reports suggest roles for bone morphogenetic proteins (BMPs) in early stages of folliculogenesis. A role for the type I BMP receptor
BmprIB
as a regulator of ovulation rates in sheep has been described recently, but little is known about the roles of BMP signaling pathways in other aspects of reproductive function. We report here that BMPRIB is essential for multiple aspects of female fertility. Mice deficient in
BmprIB
exhibit irregular estrous cycles and an impaired pseudopregnancy response.
BmprIB
mutants produce oocytes that can be fertilized
in vitro
, but defects in cumulus expansion prevent fertilization
in vivo
. This defect is associated with decreased levels of aromatase production in granulosa cells. Unexpectedly, levels of mRNA for cyclooxygenase 2, an enzyme required for cumulus expansion, are increased.
BmprIB
mutants also exhibit a failure in endometrial gland formation. The expression of
BmprIB
in uterine linings suggests that these defects are a direct consequence of loss of BMP signaling in this tissue. In summary, these studies demonstrate the importance of BMP signaling pathways for estrus cyclicity, estradiol biosynthesis, and cumulus cell expansion
in vivo
and reveal sites of action for BMP signaling pathways in reproductive tissues.
Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex
Authors: Mah Angie S. , Jang Joanne , Deshaies Raymond J.
Published: July 2002

Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14. Cdc14 activity, in turn, is regulated by a group of proteins, the mitotic exit network (MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1. The direct biochemical interactions between the components of the MEN remain largely unresolved. Here, we investigate the mechanisms that underlie activation of the protein kinase Dbf2. Dbf2 kinase activity depended on Tem1, Cdc15, and Mob1
in vivo
.
In vitro
, recombinant protein kinase Cdc15 activated recombinant Dbf2, but only when Dbf2 was bound to Mob1. Conserved phosphorylation sites Ser-374 and Thr-544 (present in the human,
Caenorhabditis elegans,
and
Drosophila melanogaster
relatives of Dbf2) were required for
DBF2
function
in vivo
, and activation of Dbf2-Mob1 by Cdc15
in vitro
. Although Cdc15 phosphorylated Dbf2, Dbf2–Mob1, and Dbf2(S374A/T544A)–Mob1, the pattern of phosphate incorporation into Dbf2 was substantially altered by either the S374A T544A mutations or omission of Mob1. Thus, Cdc15 promotes the exit from mitosis by directly switching on the kinase activity of Dbf2. We propose that Mob1 promotes this activation process by enabling Cdc15 to phosphorylate the critical Ser-374 and Thr-544 phosphoacceptor sites of Dbf2.
Genes linked by fusion events are generally of the same functional category: A systematic analysis of 30 microbial genomes
Authors: Yanai Itai , Derti Adnan , DeLisi Charles
Published: July 2002

Recent work in computational genomics has shown that a functional association between two genes can be derived from the existence of a fusion of the two as one continuous sequence in another genome. For each of 30 completely sequenced microbial genomes, we established all such fusion links among its genes and determined the distribution of links within and among 15 broad functional categories. We found that 72% of all fusion links related genes of the same functional category. A comparison of the distribution of links to simulations on the basis of a random model further confirmed the significance of intracategory fusion links. Where a gene of annotated function is linked to an unclassified gene, the fusion link suggests that the two genes belong to the same functional category. The predictions based on fusion links are shown here for
Methanobacterium thermoautotrophicum
, and another 661 predictions are available at
http://fusion.bu.edu
.
Dopamine D
1
and adenosine A
1
receptors form functionally interacting heteromeric complexes
Authors: Ginés Silvia , Hillion Joëlle , Torvinen Maria , Le Crom Stèphane , Casadó Vicent , Canela Enric I. , Rondin Sofia , Lew Jow Y. , Watson Stanley , Zoli Michele , Agnati Luigi Francesco , Vernier Philippe , Lluis Carmen , Ferré Sergi , Fuxe Kjell , Franco Rafael
Published: July 2002

The possible molecular basis for the previously described antagonistic interactions between adenosine A
1
receptors (A
1
R) and dopamine D
1
receptors (D
1
R) in the brain have been studied in mouse fibroblast Ltk
−
cells cotransfected with human A
1
R and D
1
R cDNAs or with human A
1
R and dopamine D
2
receptor (long-form) (D
2
R) cDNAs and in cortical neurons in culture. A
1
R and D
1
R, but not A
1
R and D
2
R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A
1
R/D
1
R heteromerization disappeared after pretreatment with the D
1
R agonist, but not after combined pretreatment with D
1
R and A
1
R agonists. A high degree of A
1
R and D
1
R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A
1
R and D
2
R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A
1
R agonist caused coclustering (coaggregation) of A
1
R and D
1
R, which was blocked by combined pretreatment with the D
1
R and A
1
R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D
1
R and A
1
R agonists, but not with either one alone, substantially reduced the D
1
R agonist-induced accumulation of cAMP. The A
1
R/D
1
R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A
1
R of D
1
R receptor signaling in the brain. The persistence of A
1
R/D
1
R heteromerization seems to be essential for the blockade of A
1
R agonist-induced A
1
R/D
1
R coclustering and for the desensitization of the D
1
R agonist-induced cAMP accumulation seen on combined pretreatment with D
1
R and A
1
R agonists, which indicates a potential role of A
1
R/D
1
R heteromers also in desensitization mechanisms and receptor trafficking.
“Electronic nose” detects major histocompatibility complex-dependent prerenal and postrenal odor components
Authors: Montag Stefanie , Frank Michael , Ulmer Heiko , Wernet Dorothee , Göpel Wolfgang , Rammensee Hans-Georg
Published: July 2002

Mice prefer to mate with individuals expressing different MHC genes from their own. Volatile components presenting MHC-dependent odor types are present in urine and can be detected by mice, as shown by extensive behavioral studies. Similar odor types are suspected to influence human behavior as well. Although a recent report indicates that MHC expression influences the ratio of volatile compounds such as phenylacetic acid, so far no other means than studying the behavior of mice or rats has been available to assess odor types. Here, we report the ability of a gas sensor array (referred to as “electronic nose”) to detect MHC-dependent odor types. The electronic nose consists of an array of chemophysical detectors, in our case quartz crystal microbalances and semiconducting metal-oxide sensors that change frequency or conductivity upon binding of very small numbers of individual molecules present in the gas phase of odorous fluids. The pattern of changes is characteristic for a particular smell. Our electronic nose distinguishes the urine odor types of MHC congenic mouse strains, MHC class I mutant mice, and HLA-A2 transgenic mice. In addition, MHC-dependent odor types can be detected in serum. The device also clearly differentiates between individual odor types of human sera from HLA homozygous individuals; however, HLA expression seems to have only a secondary influence. Thus, odor-type research can now be carried out with an objective and fast through-put system independent of behavioral studies.
An immune response manifested by the common occurrence of annexins I and II autoantibodies and high circulating levels of IL-6 in lung cancer
Authors: Brichory Franck M. , Misek David E. , Yim Anne-Marie , Krause Melissa C. , Giordano Thomas J. , Beer David G. , Hanash Samir M.
Published: July 2002

The identification of circulating tumor antigens or their related
autoantibodies provides a means for early cancer diagnosis as well as
leads for therapy. The purpose of this study was to identify proteins
that commonly induce a humoral response in lung cancer by using a
proteomic approach and to investigate biological processes that may be
associated with the development of autoantibodies. Aliquots of
solubilized proteins from a lung adenocarcinoma cell line (A549) and
from lung tumors were subjected to two-dimensional PAGE, followed by
Western blot analysis in which individual sera were tested for primary
antibodies. Sera from 54 newly diagnosed patients with lung cancer and
60 patients with other cancers and from 61 noncancer controls were
analyzed. Sera from 60% of patients with lung adenocarcinoma and 33%
of patients with squamous cell lung carcinoma but none of the noncancer
controls exhibited IgG-based reactivity against proteins identified as
glycosylated annexins I and/or II. Immunohistochemical analysis
showed that annexin I was expressed diffusely in neoplastic cells in
lung tumor tissues, whereas annexin II was predominant at the cell
surface. Interestingly, IL-6 levels were significantly higher in sera
of antibody-positive lung cancer patients compared with
antibody-negative patients and controls. We conclude that an immune
response manifested by annexins I and II autoantibodies occurs commonly
in lung cancer and is associated with high circulating levels of an
inflammatory cytokine. The proteomic approach we have implemented has
utility for the development of serum-based assays for cancer diagnosis
as we report in this paper on the discovery of antiannexins I and/or
II in sera from patients with lung cancer.
Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs
Authors: Yang Jing , Wu Jie , Kowalska M. Anna , Dalvi Ashutosh , Prevost Nicolas , O'Brien Peter J. , Manning David , Poncz Mortimer , Lucki Irwin , Blendy Julie A. , Brass Lawrence F.
Published: July 2002

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gzis a member of the Gifamily of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the α subunit of Gzin mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gzαalso results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Giαfamily members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.
Building a dictionary for genomes: Identification of presumptive regulatory sites by statistical analysis
Authors: Bussemaker Harmen J. , Li Hao , Siggia Eric D.
Published: July 2002

The availability of complete genome sequences and mRNA expression data for all genes creates new opportunities and challenges for identifying DNA sequence motifs that control gene expression. An algorithm, “MobyDick,” is presented that decomposes a set of DNA sequences into the most probable dictionary of motifs or words. This method is applicable to any set of DNA sequences: for example, all upstream regions in a genome or all genes expressed under certain conditions. Identification of words is based on a probabilistic segmentation model in which the significance of longer words is deduced from the frequency of shorter ones of various lengths, eliminating the need for a separate set of reference data to define probabilities. We have built a dictionary with 1,200 words for the 6,000 upstream regulatory regions in the yeast genome; the 500 most significant words (some with as few as 10 copies in all of the upstream regions) match 114 of 443 experimentally determined sites (a significance level of 18 standard deviations). When analyzing all of the genes up-regulated during sporulation as a group, we find many motifs in addition to the few previously identified by analyzing the subclusters individually to the expression subclusters. Applying MobyDick to the genes derepressed when the general repressor Tup1 is deleted, we find known as well as putative binding sites for its regulatory partners.
Delivery of the Cre recombinase by a self-deleting lentiviral vector: Efficient gene targeting
in vivo
Authors: Pfeifer Alexander , Brandon Eugene P. , Kootstra Neeltje , Gage Fred H. , Verma Inder M.
Published: July 2002

The Cre recombinase (Cre) from bacteriophage P1 is an important
tool for genetic engineering in mammalian cells. We constructed
lentiviral vectors that efficiently deliver Cre
in vitro
and
in vivo
. Surprisingly, we found a significant
reduction in proliferation and an accumulation in the
G
2
/M phase of Cre-expressing cells. To minimize the toxic
effect of Cre, we designed a lentiviral vector that integrates into the
host genome, expresses Cre in the target cell, and is subsequently
deleted from the genome in a Cre-dependent manner. Thus, the activity
of Cre terminates its own expression (self-deleting). We showed
efficient modification of target genes
in vitro
and in
the brain after transduction with the self-deleting vectors. In
contrast to sustained Cre expression, transient expression of Cre from
the self-deleting vector induced significantly less cytotoxicity. Such
a self-deleting Cre vector is a promising tool for the induction of
conditional gene modifications with minimal Cre toxicity
in
vivo
.