bluuebunny/crossref_metadata_2025 huggingface dataset:
G protein-coupled receptor kinase 4 gene variants in human essential hypertension
Authors: Felder Robin A. , Sanada Hironobu , Xu Jing , Yu Pei-Ying , Wang Zheng , Watanabe Hidetsuna , Asico Laureano D. , Wang Wei , Zheng Shaopeng , Yamaguchi Ikuyo , Williams Scott M. , Gainer James , Brown Nancy J. , Hazen-Martin Debra , Wong Lee-Jun C. , Robillard Jean E. , Carey Robert M. , Eisner Gilbert M. , Jose Pedro A.
Published: July 2002

Essential hypertension has a heritability as high as 30–50%, but its genetic cause(s) has not been determined despite intensive investigation. The renal dopaminergic system exerts a pivotal role in maintaining fluid and electrolyte balance and participates in the pathogenesis of genetic hypertension. In genetic hypertension, the ability of dopamine and D
1
-like agonists to increase urinary sodium excretion is impaired. A defective coupling between the

D
1

dopamine receptor and the G protein/effector enzyme complex in the proximal tubule of the kidney is the cause of the impaired renal dopaminergic action in genetic rodent and human essential hypertension. We now report that, in human essential hypertension, single nucleotide polymorphisms of a G protein-coupled receptor kinase,
GRK4
γ, increase G protein-coupled receptor kinase (GRK) activity and cause the serine phosphorylation and uncoupling of the D
1
receptor from its G protein/effector enzyme complex in the renal proximal tubule and in transfected Chinese hamster ovary cells. Moreover, expressing
GRK4
γ
A142V
but not the wild-type gene in transgenic mice produces hypertension and impairs the diuretic and natriuretic but not the hypotensive effects of D
1
-like agonist stimulation. These findings provide a mechanism for the D
1
receptor coupling defect in the kidney and may explain the inability of the kidney to properly excrete sodium in genetic hypertension.
One of the two cytoplasmic actin isoforms in
Drosophila
is essential
Authors: Wagner Cynthia R. , Mahowald Anthony P. , Miller Kathryn G.
Published: July 2002

Actin is a highly conserved protein found in all eukaryotic organisms. Most organisms have multiple cytoplasmic actin genes that encode isoforms with slightly different amino acid sequences. These different isoforms are coexpressed in many cell types. Why organisms have multiple very similar cytoplasmic actin genes is unclear. We have addressed this question with the cytoplasmic actins in
Drosophila
, Act5C, and Act42A. These isoforms differ by only two amino acids and both genes are expressed in all cells at all times during development. We identified
P
element insertions in the
Act5C
gene that resulted in a lethal phenotype. The lethal phenotype is rescued by a transgene with a genomic fragment that includes
Act5C
regulatory and amino acid coding sequences. A hybrid transgene containing the protein coding sequence for the Act42A isoform, under the control of the regulatory regions of the
Act5C
gene, also rescues the lethality of the
Act5C
mutants. Furthermore, flies that carry only one copy each of
Act5C
and
Act42A
are viable. These results suggest the amino acid differences between these two cytoplasmic actin isoforms are not important for function and the need for increased gene dosage to provide more actin is not likely to explain the existence of multiple genes. Instead, our results suggest that regulated expression of
Act5C
is essential to the fly.
Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress
Authors: Sobol Robert W. , Watson David E. , Nakamura Jun , Yakes F. Michael , Hou Esther , Horton Julie K. , Ladapo Joseph , Van Houten Bennett , Swenberg James A. , Tindall Kenneth R. , Samson Leona D. , Wilson Samuel H.
Published: July 2002

The long-term effect of exposure to DNA alkylating agents is entwined with the cell's genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this “gene–environment interaction” paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA β-polymerase (β-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene–environment interaction. The β-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in β-pol null cells, but not in isogenic wild-type cells; UV light exposure or
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in β-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in β-pol null cells eliminates the effect. In contrast, β-pol/AAG double null cells are slightly more mutable than the β-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure.
Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation
Authors: McEwen Bruce , Akama Keith , Alves Stephen , Brake Wayne G. , Bulloch Karen , Lee Susan , Li Chenjian , Yuen Genevieve , Milner Teresa A.
Published: July 2002

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry.
N
-methyl-
d
-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [
3
H]estrogen and [
125
I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.
Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin β subunits
Authors: Takagi Junichi , Beglova Natalia , Yalamanchili Padmaja , Blacklow Stephen C. , Springer Timothy A.
Published: July 2002

Integrin β subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999)Genomics56, 169–178]. N-terminally truncated β2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a β2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.
Manipulation of the phenolic chemistry of willows by gall-inducing sawflies
Authors: Nyman Tommi , Julkunen-Tiitto Riitta
Published: July 2002

The ability to induce galls on plants has evolved independently in
many insect orders, but the adaptive significance and evolutionary
consequences of gall induction are still largely unknown. We studied
these questions by analyzing the concentrations of various plant
defense compounds in willow leaves and sawfly galls. We found that the
galls are probably nutritionally beneficial for the sawfly larvae,
because the concentrations of most defensive phenolics are
substantially lower in gall interiors than in leaves. More importantly,
changes in chemistry occur in a similar coordinated pattern in all
studied willow species, which suggests that the insects control the
phenolic biosynthesis in their hosts. The resulting convergence of the
chemical properties of the galls both within and between host species
indicates that the role of plant chemistry in the evolution of host
shifts may be fundamentally less significant in gallers than in other
phytophagous insects.
Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.
Authors: Yaffee M , Walter P , Richter C , Müller M
Published: July 2002

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands.
A candidate live inactivatable attenuated vaccine for AIDS.
Authors: Chakrabarti B K , Maitra R K , Ma X Z , Kestler H W
Published: July 2002

The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.
Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila.
Authors: Dushay M S , Asling B , Hultmark D
Published: July 2002

NF-kappa B/Rel transcription factors are central regulators of mammalian immunity and are also implicated in the induction of cecropins and other antibacterial peptides in insects. We identified the gene for Relish, a compound Drosophila protein that, like mammalian p105 and p100, contains both a Rel homology domain and an I kappa B-like domain. Relish is strongly induced in infected flies, and it can activate transcription from the Cecropin A1 promoter. A Relish transcript is also detected in early embryos, suggesting that it acts in both immunity and embryogenesis. The presence of a compound Rel protein in Drosophila indicates that similar proteins were likely present in primordial immune systems and may serve unique signaling functions.
Transposition of DNase hypersensitive chromatin to the nuclear periphery coincides temporally with nerve growth factor-induced up-regulation of gene expression in PC12 cells.
Authors: Park P C , De Boni U
Published: July 2002

To test the hypothesis that the nonrandom organization of the contents of interphase nuclei represents a compartmentalization of function, we examined the relative, spatial relationship of small nuclear ribonucleoproteins (snRNPs) and of DNase I hypersensitive chromatin (DHC) in rat pheochromocytoma cells. In controls, DHC and snRNPs colocalized as pan-nuclear speckles. During nerve growth factor-induced differentiation, both snRNPs and DHC migrated to the nuclear periphery with the migration of DHC preceding that of snRNPs, resulting in their transient separation. The formation of DHC shells temporally coincided with an up-regulation of neurofilament light chain mRNA. This indicates that the expression of this sequence may be associated with its spatial transposition to the nuclear periphery.
Purification of the β-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells
Authors: Marshak Sonya , Totary Hana , Cerasi Erol , Melloul Danielle
Published: July 2002

The β cell-specific glucose-sensitive factor (GSF), which binds
the A3 motif of the rat I and human insulin promoters, is modulated by
extracellular glucose. A single mutation in the GSF binding site of the
human insulin promoter abolishes the stimulation by high glucose only
in normal islets, supporting the suggested physiological role of GSF in
the glucose-regulated expression of the insulin gene. GSF binding
activity was observed in all insulin-producing cells. We have therefore
purified this activity from the rat insulinoma RIN and found that a
single polypeptide of 45 kDa was responsible for DNA binding. Its amino
acid sequence, determined by microsequencing, provided direct evidence
that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as
PDX-1) and that, in addition to its essential roles in development and
differentiation of pancreatic islets and in β cell-specific gene
expression, it functions as mediator of the glucose effect on insulin
gene transcription in differentiated β cells. The human cDNA coding
for GSF/IPF-1 has been cloned, its cell and tissue distribution is
described. Its expression in the glucagon-producing cell line αTC1
transactivates the wild-type human insulin promoter more efficiently
than the mutated construct. It is demonstrated that high levels of
ectopic GSF/IPF-1 inhibit the expression of the human insulin gene in
normal islets, but not in transformed βTC1 cells. These results
suggest the existence of a control mechanism, such as requirement for a
coactivator of GSF/IPF-1, which may be present in limiting amounts in
normal as opposed to transformed β cells.
A cell cycle-regulated bacterial DNA methyltransferase is essential for viability.
Authors: Stephens C , Reisenauer A , Wright R , Shapiro L
Published: July 2002

The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.
Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate.
Authors: Afonina I , Kutyavin I , Lukhtanov E , Meyer R B , Gamper H
Published: July 2002

A minor groove binder (MGB) derivative (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate tripeptide; CDPI3) was covalently linked to the 5' or 3' end of several oligodeoxyribonucleotides (ODNs) totally complementary or possessing a single mismatch to M13mp19 single-stranded DNA. Absorption thermal denaturation and slot-blot hybridization studies showed that conjugation of CDPI3 to these ODNs increased both the specificity and the strength with which they hybridized. Primer extension of the same phage DNA by a modified form of phage T7 DNA polymerase (Sequenase) was physically blocked when a complementary 16-mer with a conjugated 5'-CDPI3 moiety was hybridized to a downstream site. Approximately 50% of the replicating complexes were arrested when the blocking ODN was equimolar to the phage DNA. Inhibition was unaffected by 3'-capping of the ODN with a hexanol group or by elimination of a preannealing step. Blockage was abolished when a single mismatch was introduced into the ODN or when the MGB was either removed or replaced by a 5'-acridine group. A 16-mer with a 3'-CDPI3 moiety failed to arrest primer extension, as did an unmodified 32-mer. We attribute the exceptional stability of hybrids formed by ODNs conjugated to a CDPI3 to the tethered tripeptide binding in the minor groove of the hybrid. When that group is linked to the 5' end of a hybridized ODN, it probably blocks DNA synthesis by inhibiting strand displacement. These ODNs conjugated to CDPI3 offer attractive features as diagnostic probes and antigene agents.
Analysis of metabolic pathways by the growth of cells in the presence of organic solvents.
Authors: Spinnler H E , Ginies C , Khan J A , Vulfson E N
Published: July 2002

A new approach to the analysis of metabolic pathways involving poorly water-soluble intermediates is proposed. It relies upon the ability of the hydrophobic intermediates formed by a sequence of intracellular reactions to cross the membrane(s) and partition between aqueous and organic phases, when cells are incubated in the presence of a nonpolar and nontoxic organic solvent. As a result of this thermodynamically driven efflux of the formed intermediates from the cell, they accumulate in the organic medium in sufficient quantities for GC-MS analysis and identification. This enables direct determination of the sequence of chemical reactions involved with no requirement for the isolation of each individual metabolite from a cell-free extract. The feasibility of the proposed methodology has been demonstrated by the elucidation of the biosynthesis of (R)-gamma-decalactone from (R)-ricinoleic acid catalyzed by the yeast Sporidiobolus ruinenii grown in the presence of decane. The corresponding 4-hydroxy-acid intermediates, formed in the course of beta-oxidation of (R)-ricinoleic acid, were simultaneously observed in a single experiment on the same chromatogram. Potential applications of this proposed methodology are briefly discussed.
Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment.
Authors: Yin D X , Schimke R T
Published: July 2002

To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation.
The use of spreading depression waves for acute and long-term monitoring of the penumbra zone of focal ischemic damage in rats.
Authors: Koroleva V I , Bures J
Published: July 2002

Slow potential recording was used for long-term monitoring of the penumbra zone surrounding an ischemic region produced by middle cerebral artery (MCA) occlusion in adult hooded rats (n = 32). Four capillary electrodes (El-E4) were chronically implanted at 2-mm intervals from AP -3, L 2 (El) to AP 0, L 5 (E4). Spontaneous or evoked slow potential waves of spreading depression (SD) were recorded during and 4 h after a 1-h MCA occlusion and at 2- to 3-day intervals afterward for 3 weeks. Duration of the initial focal ischemic depolarization was maximal at E4 and decreased with distance from the focus. SD waves in the penumbra zone were high at El and E2, low and prolonged at E3, and almost absent at E4. Amplitude of elicited SD waves was further reduced 3 days later and slowly increased in the following week. Cortical areas displaying marked reduction of SD waves in the first days after MCA occlusion either remained low or showed substantial (60%) recovery, the probability of which decreased with the duration of the initial focal ischemic depolarization and increased with the distance from the focus. It is concluded that the outcome of ischemia monitored by long-term SD recovery in the perifocal region can be partly predicted from the acute signs of MCA occlusion.
Peyer’s patch organogenesis is intact yet formation of B lymphocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kDa receptor
Authors: Pasparakis Manolis , Alexopoulou Lena , Grell Matthias , Pfizenmaier Klaus , Bluethmann Horst , Kollias George
Published: July 2002

Targeted inactivation of genes in the tumor necrosis factor (TNF)/lymphotoxin (LT) ligand and receptor system has recently revealed essential roles for these molecules in lymphoid tissue development and organization. Lymphotoxin-αβ (LTαβ)/lymphotoxin-β receptor (LTβ-R) signaling is critical for the organogenesis of lymph nodes and Peyer’s patches and for the structural compartmentalization of the splenic white pulp into distinct B and T cell areas and marginal zones. Moreover, an essential role has been demonstrated for TNF/p55 tumor necrosis factor receptor (p55TNF-R) signaling in the formation of splenic B lymphocyte follicles, follicular dendritic cell networks, and germinal centers. In contrast to a previously described essential role for the p55TNF-R in Peyer’s patch organogenesis, we show in this report that Peyer’s patches are present in both TNF and p55TNF-R knockout mice, demonstrating that these molecules are not essential for the organogenesis of this lymphoid organ. Furthermore, we show that in the absence of TNF/p55TNF-R signaling, lymphocytes segregate normally into T and B cell areas and a normal content and localization of dendritic cells is observed in both lymph nodes and Peyer’s patches. However, although B cells are found to home normally within Peyer’s patches and in the outer cortex area of lymph nodes, organized follicular structures and follicular dendritic cell networks fail to form. These results show that in contrast to LTαβ signaling, TNF signaling through the p55TNF-R is not essential for lymphoid organogenesis but rather for interactions that determine the cellular and structural organization of B cell follicles in all secondary lymphoid tissues.
Angiotensin II type
1a
receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload
Authors: Herzig Thomas C. , Jobe Shawn M. , Aoki Hiroki , Molkentin Jeffery D. , Cowley Allen W. , Izumo Seigo , Markham Bruce E.
Published: July 2002

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type
1a
receptor (AT
1a
R)-dependent mechanism. To understand how the level of AT
1a
Rs is altered in this pathological state, we studied the expression of an injected AT
1a
R promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the α-skeletal actin promoter 1.7-fold whereas the α-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT
1a
R promoter by 1.6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT
1a
R promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT
1a
R promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.
In vivo
kinetics of a redox-regulated transcriptional switch
Authors: Ding Huangen , Demple Bruce
Published: July 2002

SoxR is a transcription activator governing a cellular response to superoxide and nitric oxide in
Escherichia coli
. SoxR protein is a homodimer, and each monomer has a redox-active [2Fe–2S] cluster. Oxidation and reduction of the [2Fe–2S] clusters can reversibly activate and inactivate SoxR transcriptional activity. Here, we use electron paramagnetic resonance spectroscopy to follow the redox-switching process of SoxR protein
in vivo
. SoxR [2Fe–2S] clusters were in the fully reduced state during normal aerobic growth, but were completely oxidized after only 2-min aerobic exposure of the cells to superoxide-generating agents such as paraquat. The oxidized SoxR [2Fe–2S] clusters were rapidly re-reduced
in vivo
once the oxidative stress was removed. The
in vivo
kinetics of SoxR [2Fe–2S] cluster oxidation and reduction exactly paralleled the increase and decrease of transcription of
soxS
, the target gene for SoxR. The kinetic analysis also revealed that an oxidative stress-linked decrease in
soxS
mRNA stability contributes to the rapid attainment of a new steady state after SoxR activation. Such a redox stress-related change in
soxS
mRNA stability may represent a new level of biological control.
Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway
Authors: Suzue Kimiko , Zhou Xianzheng , Eisen Herman N. , Young Richard A.
Published: July 2002

Mice immunized with heat shock proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8 cytotoxic T lymphocytes (CTL) that recognize donor cell peptides in association with the major histocompatibility complex (MHC) class I proteins of the responding mouse. The CTL are induced apparently because peptides noncovalently associated with the isolated hsp molecules can enter the MHC class I antigen processing pathway of professional antigen-presenting cells. Using a recombinant heat shock fusion protein with a large fragment of ovalbumin covalently linked to mycobacterial hsp70, we show here that when the soluble fusion protein was injected without adjuvant into H-2bmice, CTL were produced that recognized an ovalbumin-derived peptide, SIINFEKL, in association with Kb. The peptide is known to arise from natural processing of ovalbumin in H-2bmouse cells, and CTL from the ovalbumin-hsp70-immunized mice and a highly effective CTL clone (4G3) raised against ovalbumin-expressing EL4 tumor cells (EG7-OVA) were equally effective in terms of the concentration of SIINFEKL required for half-maximal lysis in a CTL assay. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHC-disparate individuals.
Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae
Authors: Liu Pingsheng , Ying Yun-shu , Anderson Richard G. W.
Published: July 2002

The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete. Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts. Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae. The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.
The location of the cochlear amplifier: Spatial representation of a single tone on the guinea pig basilar membrane
Authors: Russell I. J. , Nilsen K. E.
Published: July 2002

Acoustic stimulation vibrates the cochlear basilar membrane, initiating a wave of displacement that travels toward the apex and reaches a peak over a restricted region according to the stimulus frequency. In this characteristic frequency region, a tone at the characteristic frequency maximally excites the sensory hair cells of the organ of Corti, which transduce it into electrical signals to produce maximum activity in the auditory nerve. Saturating, nonlinear, feedback from the motile outer hair cells is thought to provide electromechanical amplification of the travelling wave. However, neither the location nor the extent of the source of amplification, in relation to the characteristic frequency, are known. We have used a laser–diode interferometer to measure
in vivo
the distribution along the basilar membrane of nonlinear, saturating vibrations to 15 kHz tones. We estimate that the site of amplification for the 15 kHz region is restricted to a 1.25 mm length of basilar membrane centered on the 15 kHz place.
Host cell dependence of viral morphology
Authors: Roberts Paul C. , Compans Richard W.
Published: July 2002

The morphology of influenza virions was found to depend on cellular determinants. Influenza viral filaments up to 30 μm in length were observed to form at high levels on surfaces of various polarized epithelial cell types infected with the A/Udorn/72 virus. In contrast, virions produced by nonpolarized cell types infected with this virus were almost exclusively of spherical morphology. Disruption of the actin microfilament array by cytochalasin D treatment of polarized MDCK cells had a profound effect on viral morphology. Although virus titers and release of spherical particles were not reduced in the presence of cytochalasin D, we observed a 15-fold reduction in the release of filamentous particles. In contrast, the ratio of filaments to spheres produced by infected MDCK cells was not altered by the microtubule-disrupting agent nocodazole. These observations indicate that the polarized cell phenotype and the integrity of the actin microfilament network are important cellular determinants of the morphology of a filamentous influenza virus.
Abolition of morphine-immunosuppression in mice lacking the μ-opioid receptor gene
Authors: Gavériaux-Ruff Claire , Matthes Hans W. D. , Peluso Jean , Kieffer Brigitte L.
Published: July 2002

Opiates are potent analgesic and addictive compounds. They also act on immune responses, and morphine, the prototypic opiate, has been repeatedly described as an immunosuppressive drug. Pharmacological studies have suggested that the inhibitory action of opiates on immunity is mediated by multiple opioid receptor sites but molecular evidence has remained elusive. Recently, three genes encoding μ- (MOR), δ-, and κ-opioid receptors have been cloned. To investigate whether the μ-opioid receptor is functionally implicated in morphine immunosuppressionin vivo, we have examined immune responses of mice with a genetic disruption of the MOR gene. In the absence of drug, there was no difference between wild-type and mutant mice with regard to a large number of immunological endpoints, suggesting that the lack of MOR-encoded protein has little consequence on immune status. Chronic morphine administration induced lymphoid organ atrophy, diminished the ratio of CD4+CD8+cells in the thymus and strongly reduced natural killer activity in wild-type mice. None of these effects was observed in MOR-deficient mice after morphine treatment. This demonstrates that the MOR gene product represents a major molecular target for morphine action on the immune system. Because our previous studies of MOR-deficient mice have shown that this receptor protein is also responsible for morphine analgesia, reward, and physical dependence, the present results imply that MOR-targeted therapeutic drugs that are developed for the treatment of pain or opiate addiction may concomitantly influence immune responses.
Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors
Authors: Lissin Dmitri V. , Gomperts Stephen N. , Carroll Reed C. , Christine Chadwick W. , Kalman Daniel , Kitamura Marina , Hardy Stephen , Nicoll Roger A. , Malenka Robert C. , von Zastrow Mark
Published: July 2002

Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (
N
-methyl-
d
-aspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the γ-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.
The Aγ-globin 3′ element provides no unique function(s) for human β-globin locus gene regulation
Authors: Liu Qinghui , Tanimoto Keiji , Bungert Jörg , Engel James Douglas
Published: July 2002

The human β-globin locus is activated transcriptionally by a complex series of events that culminate in appropriate temporal and tissue-specific control over five separate genes during embryonic and early postnatal development. One cis-regulatory element in the locus, originally identified as an enhancer 3′ to the Aγ-globin gene, more recently has been suggested to harbor alternative or additional properties, including stage-specific silencer, insulator, nuclear matrix, or chromosome scaffold attachment activities. We have re-evaluated the activity during erythropoiesis that is conferred by this element by deleting it from a yeast artificial chromosome (YAC) containing the entire human β-globin locus and then assaying for the expression of each gene at each developmental stage after incorporation of the mutant YAC into the mouse germline. The data show that loss of the Aγ-globin 3′ element confers no phenotype in six independent lines of intact YAC mutant transgenic mice, thus demonstrating (minimally) that any activities attributable to this element are fully compensated by other DNA sequences within the β-globin locus.
Identification and characterization of
LMO
4
, an LMO gene with a novel pattern of expression during embryogenesis
Authors: Kenny Daryn A. , Jurata Linda W. , Saga Yumiko , Gill Gordon N.
Published: July 2002

LMO4
is a novel member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors.
LMO1, LMO2
, and
LMO4
have distinct expression patterns in adult tissue, and we demonstrate that nuclear retention of LMO proteins is enhanced by the nuclear LIM interactor (NLI).
In situ
hybridization to early mouse embryos of 8–14.5 days revealed a complex pattern of
LMO4
expression spatially overlapping with
NLI
and
LHX
genes.
LMO4
expression in somite is repressed in mice mutant for the segment polarity gene
Mesp2
and expanded in
Splotch
mutants. During jaw and limb outgrowth,
LMO4
and
LMO2
expression define mesenchyme that is uncommitted to regional fates. Although both
LMO2
and
LMO4
are activated in thymic blast cells, only
LMO4
is expressed in mature T cells. Mesenchymal and thymic blast cell expression patterns of
LMO4
and
LMO2
are consistent with the suggestion that LMO genes inhibit differentiation.
Expression of the Epstein–Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice
Authors: Kulwichit Wanla , Edwards Rachel Hood , Davenport Ethan M. , Baskar John F. , Godfrey Virginia , Raab-Traub Nancy
Published: July 2002

The latent membrane protein 1 (LMP1) of the Epstein–Barr virus has transforming properties in rodent fibroblasts and is expressed in most of the cancers associated with Epstein–Barr virus (EBV) infection including posttransplant lymphomas, Hodgkin’s disease, nasopharyngeal carcinoma, and AIDS-related lymphomas. In this study, three lineages of LMP1 transgenic mice were established with LMP1 expressed under the control of the Ig heavy chain promoter and enhancer. Lymphoma developed in all three lineages, and the incidence of lymphoma increased significantly with age with lymphomas developing in 42% of transgenic mice over 18 months. The expression of LMP1 was detected at high levels in the lymphoma tissues but only at trace levels in normal lymphoid tissues. Gene rearrangement of the Ig heavy chain indicated monoclonality or oligoclonality in all lymphomas, some of the lymphoid hyperplastic spleens, and some histologically normal spleens. These data reveal that LMP1, without the expression of other EBV genes, is oncogenicin vivoand indicate that LMP1 is a major contributing factor to the development of EBV-associated lymphomas.
DNA end-joining catalyzed by human cell-free extracts
Authors: Baumann Peter , West Stephen C.
Published: July 2002

Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an
in vitro
system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PK
cs
, the three subunits of the DNA-activated protein kinase DNA-PK. Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this
in vitro
system provides an important advance in the study of the mechanism of DNA end-joining in human cells.
Relationships of cereal crops and other grasses
Authors: Kellogg Elizabeth A.
Published: July 2002

The grass family includes some 10,000 species, and it encompasses
tremendous morphological, physiological, ecological, and genetic
diversity. The phylogeny of the family is becoming increasingly well
understood. There were two major radiations of grasses, an early
diversification leading to the subfamilies Pooideae, Bambusoideae, and
Oryzoideae, and a later one leading to Panicoideae, Chloridoideae,
Centothecoideae, and Arundinoideae. The phylogeny can be used to
determine the direction of changes in genome arrangement and genome
size.
Genomic sequences of aldolase C (Zebrin II) direct
lacZ
expression exclusively in non-neuronal cells of transgenic mice
Authors: Walther Ernst U. , Dichgans Martin , Maricich Stephen M. , Romito Rita R. , Yang Fei , Dziennis Suzan , Zackson Saul , Hawkes Richard , Herrup Karl
Published: July 2002

Aldolase C is regarded as the brain-specific form of fructose-1,6-bisphosphate aldolase whereas aldolase A is regarded as muscle-specific.
In situ
hybridization of mouse central nervous system using isozyme-specific probes revealed that aldolase A and C are expressed in complementary cell types. With the exception of cerebellar Purkinje cells, aldolase A mRNA is found in neurons; aldolase C message is detected in astrocytes, some cells of the pia mater, and Purkinje cells. We isolated aldolase C genomic clones that span the entire protein coding region from 1.5 kb 5′ to the transcription start site to 0.5 kb 3′ to the end of the last exon. The bacterial gene,
lacZ
, was inserted in two different locations and the constructs tested in transgenic mice. When the protein coding sequences were replaced with
lacZ
, three of five transgenic lines expressed β-galactosidase only in cells of the pia mater; one line also expressed in astrocyte-like cells. When
lacZ
was inserted into the final exon (and all structural gene sequences were retained) transgene expression was observed in astrocytes in all regions of the central nervous system as well as in pial cells. Thus, with the exception of Purkinje cell expression, the behavior of the full-length transgene mimics the endogenous aldolase C gene. The results with the shorter transgene suggest that additional enhancer elements exist within the intragenic sequences. The absence of Purkinje cell staining suggests that the
cis
elements required for this expression must be located outside of the sequences used in this study.
Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas
Authors: Miralles Francisco , Czernichow Paul , Ozaki Keiichi , Itoh Nobuyuki , Scharfmann Raphael
Published: July 2002

The development of the pancreas depends on epithelial-mesenchymal interactions. Fibroblast growth factors (FGFs) and their receptors (FGFRs 1–4) have been identified as mediators of epithelial-mesenchymal interactions in different organs. We show here that FGFR-2 IIIb and its ligands FGF-1, FGF-7, and FGF-10 are expressed throughout pancreatic development. We also show that in mesenchyme-free cultures of embryonic pancreatic epithelium FGF-1, FGF-7, and FGF-10 stimulate the growth, morphogenesis, and cytodifferentiation of the exocrine cells of the pancreas. The role of FGFs signaling through FGFR-2 IIIb was further investigated by inhibiting FGFR-2 IIIb signaling in organocultures of pancreatic explants (epithelium + mesenchyme) by using either antisense FGFR-2 IIIb oligonucleotides or a soluble recombinant FGFR-2 IIIb protein. Abrogation of FGFR-2 IIIb signaling resulted in a considerable reduction in the size of the explants and in a 2-fold reduction of the development of the exocrine cells. These results demonstrate that FGFs signaling through FGFR-2 IIIb play an important role in the development of the exocrine pancreas.
An isotopic approach for understanding the CH
3
Br budget of the atmosphere
Authors: McCauley Sean E. , Goldstein Allen H. , DePaolo Donald J.
Published: July 2002

The atmospheric budget of methyl bromide (CH
3
Br), an
ozone-depleting gas, is highly uncertain, because it has complex
sources and sinks. Although oceans, biomass burning, and industrial
production are identified as the major sources, the fraction of
CH
3
Br that is contributed by each source is not well known.
A mass-balance approach that exploits differences in the carbon
isotopic signature (δ
13
C) of CH
3
Br sources
and sinks may provide a means of reducing uncertainties in the
atmospheric budget. This approach depends on the distinctiveness of
industrially produced methyl bromide. Our δ
13
C
measurements of industrial CH
3
Br from the three largest
manufacturers worldwide yield a weighted average of −54.4‰ relative
to the Peedee Belemnite standard. This result suggests that industrial
CH
3
Br is isotopically distinct and that the carbon isotopic
composition of atmospheric CH
3
Br may indicate what fraction
of atmospheric CH
3
Br is anthropogenic.
Different circadian oscillators control Ca
2+
fluxes and
Lhcb
gene expression
Authors: Sai Jiqing , Johnson Carl Hirschie
Published: July 2002

Circadian biological clocks control many biological events, but the pathways by which these events are controlled are largely unknown. Based on a model suggesting that cytosolic-free calcium levels control the expression of the
Lhcb
gene in plants, we tested whether the circadian oscillation of free calcium is responsible for driving the rhythm of
Lhcb
expression. We found that these rhythms free-run with different periods in tobacco seedlings in constant conditions. Moreover, robust oscillations of
Lhcb
promoter activity continued in undifferentiated tobacco calli in the absence of Ca
2+
oscillations. Therefore, these two circadian rhythms are not linked hierarchically. These data provide evidence for separate circadian pacemakers controlling molecular events in plants.