bluuebunny/crossref_metadata_2025 huggingface dataset:
A dominant form of inherited retinal degeneration caused by a non-photoreceptor cell-specific mutation
Authors: Li Lei , Dowling John E.
Published: July 2002

We have isolated a dominant mutation,
night blindness a (nba)
, that causes a slow retinal degeneration in zebrafish. Heterozygous
nba
fish have normal vision through 2–3 months of age but subsequently become night blind. By 9.5 months of age, visual sensitivity of affected fish may be decreased more than two log units, or 100-fold, as measured behaviorally. Electroretinographic (ERG) thresholds of mutant fish are also raised significantly, and the ERG b-wave shows a delayed implicit time. These defects are due primarily to a late-onset photoreceptor cell degeneration involving initially the rods but eventually the cones as well. Homozygous
nba
fish display an early-onset neuronal degeneration throughout the retina and elsewhere in the central nervous system. As a result, animals develop with small eyes and die by 4–5 days postfertilization (pf). These latter data indicate that the mutation affecting
nba
fish is not in a photoreceptor cell-specific gene.
Muscarinic stimulation of synaptic activity by protein kinase C is inhibited by adenosine in cultured hippocampal neurons
Authors: Bouron Alexandre , Reuter Harald
Published: July 2002

We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1–43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca
2+
signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular Ca
2+
concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis(2-aminophenoxy)ethane-
N,N,N′,N′
-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular Ca
2+
concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4β-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.
Conservation of fibroblast growth factor function in lens regeneration
Authors: Del Rio-Tsonis Katia , Jung Jae Chang , Chiu Ing-Ming , Tsonis Panagiotis A.
Published: July 2002

In urodele amphibians, lens induction during development and regeneration occurs through different pathways. During development, the lens is induced from the mutual interaction of the ectoderm and the optic vesicle, whereas after lentectomy the lens is regenerated through the transdifferentiation of the iris-pigmented epithelial cells. Given the known role of fibroblast growth factors (FGFs) during lens development, we examined whether or not the expression and the effects of exogenous FGF during urodele lens regeneration were conserved. In this paper, we describe expression of FGF-1 and its receptors, FGFR-2 (KGFR and
bek
variants) and FGFR-3, in newts during lens regeneration. Expression of these genes was readily observed in the dedifferentiating pigmented epithelial cells, and the levels of expression were high in the lens epithelium and the differentiating fibers and lower in the retina. These patterns of expression implied involvement of FGFs in lens regeneration. To further elucidate this function, we examined the effects of exogenous FGF-1 and FGF-4 during lens regeneration. FGF-1 or FGF-4 treatment in lentectomized eyes resulted in the induction of abnormalities reminiscent to the ones induced during lens development in transgenic mice. Effects included transformation of epithelial cells to fiber cells, double lens regeneration, and lenses with abnormal polarity. These results establish that FGF molecules are key factors in fiber differentiation, polarity, and morphogenesis of the lens during regeneration even though the regenerating lens is induced by a different mechanism than in lens development. In this sense, FGF function in lens regeneration and development should be regarded as conserved. Such conservation should help elucidate the mechanisms of lens regeneration in urodeles and its absence in higher vertebrates.
Backtracking leukemia to birth: Identification of clonotypic gene fusion sequences in neonatal blood spots
Authors: Gale Karin B. , Ford Anthony M. , Repp Reinald , Borkhardt Arndt , Keller Claudia , Eden Osborn B. , Greaves Mel F.
Published: July 2002

Epidemiological evidence has suggested that some pediatric leukemias may be initiated
in utero
and, for some pairs of identical twins with concordant leukemia, this possibility has been strongly endorsed by molecular studies of clonality. Direct evidence for a prenatal origin can only be derived by prospective or retrospective detection of leukemia-specific molecular abnormalities in fetal or newborn samples. We report a PCR-based method that has been developed to scrutinize neonatal blood spots (Guthrie cards) for the presence of numerically infrequent leukemic cells at birth in individuals who subsequently developed leukemia. We demonstrate that unique or clonotypic
MLL-AF4
genomic fusion sequences are present and detectable in neonatal blood spots from individuals who were diagnosed with acute lymphoblastic leukemia at ages 5 months to 2 years and, therefore, have arisen during fetal hematopoiesis
in utero
. This result provides unequivocal evidence for a prenatal initiation of acute leukemia in young patients. The method should be applicable to other fusion genes in children with common subtypes of leukemia and will be of value in attempts to unravel the natural history and etiology of this major subtype of pediatric cancer.
The complete mitochondrial genome of the wallaroo (
Macropus robustus
) and the phylogenetic relationship among Monotremata, Marsupialia, and Eutheria
Authors: Janke Axel , Xu Xiufeng , Arnason Ulfur
Published: July 2002

The complete mitochondrial DNA (mtDNA) (16,896 nt) of the wallaroo (
Macropus robustus
) was sequenced. The concatenated amino acid sequences of 12 mitochondrial protein-coding genes of the wallaroo plus those of a number of other mammals were included in a phylogenetic study of early mammalian divergences. The analysis joined monotremes and marsupials (the Marsupionta hypothesis) to the exclusion of eutherians. The analysis rejected significantly the commonly acknowledged Theria hypothesis, according to which Marsupialia and Eutheria are grouped together to the exclusion of Monotremata. The region harboring the gene for lysine tRNA (tRNA-Lys) in the mtDNA of other vertebrates is in the wallaroo occupied by a sequence (tRNA-Lys) that lacks both an anticodon loop as well as the anticodon for the amino acid lysine. An alternative tRNA-Lys gene was not identified in any other region of the mtDNA of the wallaroo, suggesting that a tRNA-Lys of nuclear origin is imported into marsupial mitochondria. Previously described RNA editing of tRNA-Asp and rearrangement of some tRNA genes were reconfirmed in the mtDNA of the wallaroo. The divergence between Monotremata/Marsupialia and Eutheria was timed to ≈130 million years before present (MYBP). The same calculations suggested that Monotremata and Marsupialia diverged ≈115 MYBP and that Australian and American marsupials separated ≈75 MYBP. The findings also show that many, probably most, extant eutherian orders had their origin in middle to late Cretaceous times, 115–65 MYBP.
Pituitary adenylate cyclase-activating polypeptide is an autocrine inhibitor of mitosis in cultured cortical precursor cells
Authors: Lu Nairu , DiCicco-Bloom Emanuel
Published: July 2002

During brain development, an intricate array of signals is likely to control the transition from proliferation to differentiation, particularly in the complex cerebral cortex. Although factors regulating proliferation and differentiation have been identified, little is known about mechanisms governing the exit of precursors from the cell cycle. We now report that pituitary adenylate cyclase-activating polypeptide (PACAP), a new member of the vasoactive intestinal peptide family expressed in embryonic brain, promotes this transition. In virtually pure cultures of embryonic day 13.5 (E13.5) rat cortical precursors, PACAP inhibited [
3
H]thymidine incorporation by 43%, decreasing the proportion of mitotic cells. Moreover, the peptide promoted morphological and biochemical differentiation; PACAP elicited a 2-fold increase in cells bearing neurites and a 30% increase in neurotrophin
trk
B receptor expression, indicating that PACAP induced cell cycle withdrawal and promoted neuronal differentiation. The expression of PACAP ligand and receptor in precursors raised the possibility of autocrine function. Indeed, 85% of cells exhibited PACAP immunoreactivity while 64% expressed type I receptor, which, in turn, mediated cAMP activation and phosphorylated cAMP response element binding protein nuclear signaling. Furthermore, treatment with the PACAP antagonist or neutralizing antibody increased DNA synthesis and proliferation, which is consistent with interruption of ongoing mitotic inhibition mediated by endogenous PACAP. Our observations suggest that cortical precursors produce PACAP as an autocrine signal to elicit cell cycle withdrawal, inducing the transition from proliferation to neuronal differentiation.
Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors α and δ
Authors: Forman Barry Marc , Chen Jasmine , Evans Ronald M.
Published: July 2002

Fatty acids (FAs) and their derivatives are essential cellular metabolites whose concentrations must be closely regulated. This implies that regulatory circuits exist which can sense changes in FA levels. Indeed, the peroxisome proliferator-activated receptor α (PPARα) regulates lipid homeostasis and is transcriptionally activated by a variety of lipid-like compounds. It remains unclear as to how these structurally diverse compounds can activate a single receptor. We have developed a novel conformation-based assay that screens activators for their ability to bind to PPARα/δ and induce DNA binding. We show here that specific FAs, eicosanoids, and hypolipidemic drugs are ligands for PPARα or PPARδ. Because altered FA levels are associated with obesity, atherosclerosis, hypertension, and diabetes, PPARs may serve as molecular sensors that are central to the development and treatment of these metabolic disorders.
Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA
Authors: Newton Salete M. C. , Allen Jennifer S. , Cao Zhenghua , Qi Zengbiao , Jiang Xunqing , Sprencel Cathy , Igo John D. , Foster Samuel B. , Payne Marvin A. , Klebba Phillip E.
Published: July 2002

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypicEscherichia colisiderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.
The basic-helix–loop–helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors
Authors: Hogenesch John B. , Gu Yi-Zhong , Jain Sanjay , Bradfield Christopher A.
Published: July 2002

We report that MOP3 is a general dimerization partner for a subset of the basic-helix–loop–helix (bHLH)-PER–ARNT–SIM (PAS) superfamily of transcriptional regulators. We demonstrated that MOP3 interacts with MOP4, CLOCK, hypoxia-inducible factor 1α (HIF1α), and HIF2α. A DNA selection protocol revealed that the MOP3-MOP4 heterodimer bound a CACGTGA-containing DNA element. Transient transfection experiments demonstrated that the MOP3-MOP4 and MOP3-CLOCK complexes bound this element in COS-1 cells and drove transcription from a linked luciferase reporter gene. We also deduced the high-affinity DNA binding sites for MOP3-HIF1α complex (TACGTGA) and used transient transfection experiments to demonstrate that the MOP3-HIF1α and MOP3-HIF2α heterodimers bound this element, drove transcription, and responded to cellular hypoxia. Finally, we found that MOP3 mRNA expression overlaps in a number of tissues with each of its four potential partner molecules
in vivo
.
Localization of the Wilson’s disease protein product to mitochondria
Authors: Lutsenko Svetlana , Cooper Matthew J.
Published: July 2002

Wilson’s disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.
Acoustic overstimulation increases outer hair cell Ca2+concentrations and causes dynamic contractions of the hearing organ
Authors: Fridberger Anders , Flock Åke , Ulfendahl Mats , Flock Britta
Published: July 2002

The dynamic responses of the hearing organ to acoustic overstimulation were investigated using the guinea pig isolated temporal bone preparation. The organ was loaded with the fluorescent Ca2+indicator Fluo-3, and the cochlear electric responses to low-level tones were recorded through a microelectrode in the scala media. After overstimulation, the amplitude of the cochlear potentials decreased significantly. In some cases, rapid recovery was seen with the potentials returning to their initial amplitude. In 12 of 14 cases in which overstimulation gave a decrease in the cochlear responses, significant elevations of the cytoplasmic [Ca2+] in the outer hair cells were seen. [Ca2+] increases appeared immediately after terminating the overstimulation, with partial recovery taking place in the ensuing 30 min in some preparations. Such [Ca2+] changes were not seen in preparations that were stimulated at levels that did not cause an amplitude change in the cochlear potentials. The overstimulation also gave rise to a contraction, evident as a decrease of the width of the organ of Corti. The average contraction in 10 preparations was 9 μm (SE 2 μm). Partial or complete recovery was seen within 30–45 min after the overstimulation. The [Ca2+] changes and the contraction are likely to produce major functional alterations and consequently are suggested to be a factor contributing strongly to the loss of function seen after exposure to loud sounds.
A role for jasmonate in pathogen defense of
Arabidopsis
Authors: Vijayan Perumal , Shockey Jay , Lévesque C. André , Cook R. James , Browse John
Published: July 2002

To investigate the role of jasmonate in the defense of plants against fungal pathogens, we have studied a mutant of
Arabidopsis
,
fad3–2 fad7–2 fad8
, that cannot accumulate jasmonate. Mutant plants were extremely susceptible to root rot caused by the fungal root pathogen
Pythium mastophorum
(Drechs.), even though neighboring wild-type plants were largely unaffected by this fungus. Application of exogenous methyl jasmonate substantially protected mutant plants, reducing the incidence of disease to a level close to that of wild-type controls. A similar treatment with methyl jasmonate did not protect the jasmonate-insensitive mutant
coi1
from infection, showing that protective action of applied jasmonate against
P. mastophorum
was mediated by the induction of plant defense mechanisms rather than by a direct antifungal action. Transcripts of three jasmonate-responsive defense genes are induced by
Pythium
challenge in the wild-type but not in the jasmonate-deficient mutant.
Pythium
species are ubiquitous in soil and root habitats world-wide, but most (including
P. mastophorum
) are considered to be minor pathogens. Our results indicate that jasmonate is essential for plant defense against
Pythium
and, because of the high exposure of plant roots to
Pythium
inoculum in soil, may well be fundamental to survival of plants in nature. Our results further indicate that the
fad3–2 fad7–2 fad8
mutant is an appropriate genetic model for studying the role of this important signaling molecule in pathogen defense.
Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast
Authors: Hochstenbach Frans , Klis Frans M. , van den Ende Herman , van Donselaar Elly , Peters Peter J. , Klausner Richard D.
Published: July 2002

The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene,
ags1
+
, which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis.
Structure of
Fok
I has implications for DNA cleavage
Authors: Wah David A. , Bitinaite Jurate , Schildkraut Ira , Aggarwal Aneel K.
Published: July 2002

Fok
I is a member an unusual class of restriction enzymes that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence.
Fok
I consists of an N-terminal DNA recognition domain and a C-terminal cleavage domain. The bipartite nature of
Fok
I has led to the development of artificial enzymes with novel specificities. We have solved the structure of
Fok
I to 2.3 Å resolution. The structure reveals a dimer, in which the dimerization interface is mediated by the cleavage domain. Each monomer has an overall conformation similar to that found in the
Fok
I–DNA complex, with the cleavage domain packing alongside the DNA recognition domain. In corroboration with the cleavage data presented in the accompanying paper in this issue of
Proceedings
, we propose a model for
Fok
I DNA cleavage that requires the dimerization of
Fok
I on DNA to cleave both DNA strands.
Increased anxiety of mice lacking the serotonin
1A
receptor
Authors: Parks Christopher L. , Robinson Patricia S. , Sibille Etienne , Shenk Thomas , Toth Miklos
Published: July 2002

Brain serotonin (5-HT) has been implicated in a number of physiological processes and pathological conditions. These effects are mediated by at least 14 different 5-HT receptors. We have inactivated the gene encoding the 5-HT
1A
receptor in mice and found that receptor-deficient animals have an increased tendency to avoid a novel and fearful environment and to escape a stressful situation, behaviors consistent with an increased anxiety and stress response. Based on the role of the 5-HT
1A
receptor in the feedback regulation of the 5-HT system, we hypothesize that an increased serotonergic neurotransmission is responsible for the anxiety-like behavior of receptor-deficient animals. This view is consistent with earlier studies showing that pharmacological activation of the 5-HT system is anxiogenic in animal models and also in humans.
A photosystem I reaction center driven by chlorophyll
d
in oxygenic photosynthesis
Authors: Hu Qiang , Miyashita Hideaki , Iwasaki Ikuko , Kurano Norihide , Miyachi Shigetoh , Iwaki Masayo , Itoh Shigeru
Published: July 2002

A far-red type of oxygenic photosynthesis was discovered in
Acaryochloris marina
, a recently found marine prokaryote that produces an atypical pigment chlorophyll
d
(Chl
d
). The purified photosystem I reaction center complex of
A. marina
contained 180 Chl
d
per 1 Chl
a
with PsaA–F, -L, -K, and two extra polypeptides. Laser excitation induced absorption changes of reaction center Chl
d
that was named P740 after its peak wavelength. A midpoint oxidation reduction potential of P740 was determined to be +335 mV. P740 uses light of significantly low quantum energy (740 nm = 1.68 eV) but generates a reducing power almost equivalent to that produced by a special pair of Chl
a
(P700) that absorbs red light at 700 nm (1.77 eV) in photosystem I of plants and cyanobacteria. The oxygenic photosynthesis based on Chl
d
might either be an acclimation to the far-red light environments or an evolutionary intermediate between the red-absorbing oxygenic and the far-red absorbing anoxygenic photosynthesis that uses bacteriochlorophylls.
Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice
Authors: Saftig Paul , Hunziker Ernst , Wehmeyer Olaf , Jones Sheila , Boyde Alan , Rommerskirch Winfried , Moritz Jörg Detlev , Schu Peter , von Figura Kurt
Published: July 2002

Cathepsin K is a recently identified lysosomal cysteine proteinase. It is abundant in osteoclasts, where it is believed to play a vital role in the resorption and remodeling of bone. Pycnodysostosis is a rare inherited osteochondrodysplasia that is caused by mutations of the cathepsin-K gene, characterized by osteosclerosis, short stature, and acroosteolysis of the distal phalanges. With a view to delineating the role of cathepsin K in bone resorption, we generated mice with a targeted disruption of this proteinase. Cathepsin-K-deficient mice survive and are fertile, but display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space. Cathepsin-K-deficient osteoclasts manifested a modified ultrastructural appearance: their resorptive surface was poorly defined with a broad demineralized matrix fringe containing undigested fine collagen fibrils; their ruffled borders lacked crystal-like inclusions, and they were devoid of collagen-fibril-containing cytoplasmic vacuoles. Assaying the resorptive activity of cathepsin-K-deficient osteoclasts
in vitro
revealed this function to be severely impaired, which supports the contention that cathepsin K is of major importance in bone remodeling.
Cellular gene expression altered by human cytomegalovirus: Global monitoring with oligonucleotide arrays
Authors: Zhu Hua , Cong Jian-Ping , Mamtora Gargi , Gingeras Thomas , Shenk Thomas
Published: July 2002

Mechanistic insights to viral replication and pathogenesis generally have come from the analysis of viral gene products, either by studying their biochemical activities and interactions individually or by creating mutant viruses and analyzing their phenotype. Now it is possible to identify and catalog the host cell genes whose mRNA levels change in response to a pathogen. We have used DNA array technology to monitor the level of ≈6,600 human mRNAs in uninfected as compared with human cytomegalovirus-infected cells. The level of 258 mRNAs changed by a factor of 4 or more before the onset of viral DNA replication. Several of these mRNAs encode gene products that might play key roles in virus-induced pathogenesis, identifying them as intriguing targets for further study.
Role of the proteasome and NF-κB in streptococcal cell wall-induced polyarthritis
Authors: Palombella Vito J. , Conner Elaine M. , Fuseler John W. , Destree Antonia , Davis Jonathan M. , Laroux F. Stephen , Wolf Robert E. , Huang Jianqing , Brand Stephen , Elliott Peter J. , Lazarus Douglas , McCormack Teresa , Parent Lana , Stein Ross , Adams Julian , Grisham Matthew B.
Published: July 2002

The transcription factor NF-κB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-κB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IκB is degraded by the ubiquitin–proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-κB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin–proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-κB and the subsequent transcription of genes that are regulated by NF-κB. Significantly, PS-341 displayed similar effectsin vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IκBα degradation and NF-κB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin–proteasome pathway and NF-κB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.
Presence and phosphorylation of transcription factors in developing dendrites
Authors: Crino Peter , Khodakhah Kamran , Becker Kevin , Ginsberg Stephen , Hemby Scott , Eberwine James
Published: July 2002

In screening amplified poly(A) mRNA from hippocampal dendrites and growth cones in culture to determine candidates for local translation, we found that select transcription factor mRNAs were present. We hypothesized that synthesis of transcription factor proteins within dendrites would provide a direct signaling pathway between the distal dendrite and the nucleus resulting in modulation of gene expression important for neuronal differentiation. To evaluate this possibility, radiolabeled amplified antisense RNA was used to probe slot blots of transcription factor cDNAs as well as arrayed blots of zinc finger transcription factors. The mRNAs encoding the cAMP response element binding protein (CREB),
zif
268, and one putative transcription factor were detected. We expanded upon these results showing that CREB protein is present in dendrites, that translation of CREB mRNA in isolated dendrites is feasible and that CREB protein found in dendrites can interact with the cis-acting cyclic AMP reponse element DNA sequence by using an
in situ
Southwestern assay. Further, CREB protein in dendrites is not transported to this site from the cell body because fluorescently tagged CREB microperfused into the soma did not diffuse into the dendrites. In addition, CREB protein microperfused into dendrites was rapidly transported to the nucleus, its likely site of bioactivity. Lastly, by using the isolated dendrite system we show that phosphorylation of Ser-133 on CREB protein can occur in isolated dendrites independent of the nucleus. These data provide a regulatory pathway in which transcription factors synthesized and posttranslationally modified in dendrites directly alter gene expression bypassing the integration of signal transduction pathways that converge on the nucleus.
Vibro-acoustography: An imaging modality based on ultrasound-stimulated acoustic emission
Authors: Fatemi Mostafa , Greenleaf James F.
Published: July 2002

We describe theoretical principles of an imaging modality that uses the acoustic response of an object to a highly localized dynamic radiation force of an ultrasound field. In this method, named ultrasound-stimulated vibro-acoustography (USVA), ultrasound is used to exert a low-frequency (in kHz range) force on the object. In response, a portion of the object vibrates sinusoidally in a pattern determined by its viscoelastic properties. The acoustic emission field resulting from object vibration is detected and used to form an image that represents both the ultrasonic and low-frequency (kHz range) mechanical characteristics of the object. We report the relation between the emitted acoustic field and the incident ultrasonic pressure field in terms of object parameters. Also, we present the point-spread function of the imaging system. The experimental images in this report have a resolution of about 700 μm, high contrast, and high signal-to-noise ratio. USVA is sensitive enough to detect object motions on the order of nanometers. Possible applications include medical imaging and material evaluation.
The PTEN tumor suppressor homolog in
Caenorhabditis
elegans
regulates longevity and dauer formation in an insulin receptor-like signaling pathway
Authors: Mihaylova Valia T. , Borland Christina Z. , Manjarrez Laura , Stern Michael J. , Sun Hong
Published: July 2002

Inactivation of the tumor suppressor
PTEN
gene is found in a variety of human cancers and in cancer predisposition syndromes. Recently, PTEN protein has been shown to possess phosphatase activity on phosphatidylinositol 3,4,5-trisphosphate, a product of phosphatidylinositol 3-kinase. We have identified a homolog of PTEN in
Caenorhabditis
elegans
and have found that it corresponds to the
daf-18
gene, which had been defined by a single, phenotypically weak allele,
daf-18(e1375).
By analyzing an allele,
daf-18(nr2037)
, which bears a deletion of the catalytic portion of CePTEN/DAF-18, we have shown that mutation in
daf-18
can completely suppress the dauer-constitutive phenotype caused by inactivation of
daf-2
or
age-1
, which encode an insulin receptor-like molecule and the catalytic subunit of phosphatidylinositol 3-kinase, respectively. In addition,
daf-18(nr2037)
dramatically shortens lifespan, both in a wild-type background and in a
daf-2
mutant background that normally prolongs lifespan. The lifespan in a
daf-18(nr2037)
mutant can be restored to essentially that of wild type when combined with a
daf-2
mutation. Our studies provide genetic evidence that, in
C. elegans
, the PTEN homolog DAF-18 functions as a negative regulator of the DAF-2 and AGE-1 signaling pathway, consistent with the notion that DAF-18 acts a phosphatidylinositol 3,4,5-trisphosphate phosphatase
in vivo
. Furthermore, our studies have uncovered a longevity-promoting activity of the PTEN homolog in
C. elegans
.
Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects
Authors: Sheikh Kazim A. , Sun Ji , Liu Yujing , Kawai Hiromichi , Crawford Thomas O. , Proia Richard L. , Griffin John W. , Schnaar Ronald L.
Published: July 2002

Gangliosides are a family of sialic acid-containing glycosphingolipids highly enriched in the mammalian nervous system. Although they are the major sialoglycoconjugates in the brain, their neurobiological functions remain poorly defined. By disrupting the gene for a key enzyme in complex ganglioside biosynthesis (GM2/GD2 synthase; EC
2.4.1.92
) we generated mice that express only simple gangliosides (GM3/GD3) and examined their central and peripheral nervous systems. The complex ganglioside knockout mice display decreased central myelination, axonal degeneration in both the central and peripheral nervous systems, and demyelination in peripheral nerves. The pathological features of their nervous system closely resemble those reported in mice with a disrupted gene for myelin-associated glycoprotein (MAG), a myelin receptor that binds to complex brain gangliosides
in vitro
. Furthermore, GM2/GD2 synthase knockout mice have reduced MAG expression in the central nervous system. These results indicate that complex gangliosides function in central myelination and maintaining the integrity of axons and myelin. They also support the theory that complex gangliosides are endogenous ligands for MAG. The data extend and clarify prior observations on a similar mouse model, which reported only subtle conduction defects in their nervous system [Takamiya, K., Yamamoto, A., Furukawa, K., Yamashiro, S., Shin, M., Okada, M., Fukumoto, S., Haraguchi, M., Takeda, N., Fujimura, K.,
et al.
(1996)
Proc. Natl. Acad. Sci. USA
93, 10662–10667].
A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain
Authors: Porreca Frank , Lai Josephine , Bian Di , Wegert Sandra , Ossipov Michael H. , Eglen Richard M. , Kassotakis Laura , Novakovic Sanja , Rabert Douglas K. , Sangameswaran Lakshmi , Hunter John C.
Published: July 2002

Alterations in sodium channel expression and function have been suggested as a key molecular event underlying the abnormal processing of pain after peripheral nerve or tissue injury. Although the relative contribution of individual sodium channel subtypes to this process is unclear, the biophysical properties of the tetrodotoxin-resistant current, mediated, at least in part, by the sodium channel PN3 (SNS), suggests that it may play a specialized, pathophysiological role in the sustained, repetitive firing of the peripheral neuron after injury. Moreover, this hypothesis is supported by evidence demonstrating that selective “knock-down” of PN3 protein in the dorsal root ganglion with specific antisense oligodeoxynucleotides prevents hyperalgesia and allodynia caused by either chronic nerve or tissue injury. In contrast, knock-down of NaN/SNS2 protein, a sodium channel that may be a second possible candidate for the tetrodotoxin-resistant current, appears to have no effect on nerve injury-induced behavioral responses. These data suggest that relief from chronic inflammatory or neuropathic pain might be achieved by selective blockade or inhibition of PN3 expression. In light of the restricted distribution of PN3 to sensory neurons, such an approach might offer effective pain relief without a significant side-effect liability.
Brain-derived neurotrophic factor is an endogenous modulator of nociceptive responses in the spinal cord
Authors: Thompson S. W. N. , Bennett D. L. H. , Kerr B. J. , Bradbury E. J. , McMahon S. B.
Published: July 2002

The primary sensory neurons that respond to noxious stimulation and
project to the spinal cord are known to fall into two distinct groups:
one sensitive to nerve growth factor and the other sensitive to glial
cell-line-derived neurotrophic factor. There is currently considerable
interest in the ways in which these factors may regulate nociceptor
properties. Recently, however, it has emerged that another trophic
factor—brain-derived neurotrophic factor (BDNF)—may play an important
neuromodulatory role in the dorsal horn of the spinal cord. BDNF meets
many of the criteria necessary to establish it as a
neurotransmitter/neuromodulator in small-diameter nociceptive
neurons. It is synthesized by these neurons and packaged in dense core
vesicles in nociceptor terminals in the superficial dorsal horn. It is
markedly up-regulated in inflammatory conditions in a nerve growth
factor-dependent fashion. Postsynaptic cells in this region express
receptors for BDNF. Spinal neurons show increased excitability to
nociceptive inputs after treatment with exogenous BDNF. There are both
electrophysiological and behavioral data showing that antagonism of
BDNF at least partially prevents some aspects of central sensitization.
Together, these findings suggest that BDNF may be released from primary
sensory nociceptors with activity, particularly in some persistent pain
states, and may then increase the excitability of rostrally projecting
second-order systems. BDNF released from nociceptive terminals may thus
contribute to the sensory abnormalities associated with some
pathophysiological states, notably inflammatory conditions.
Time-resolved analysis and visualization of dynamic processes in living cells
Authors: Tvaruskó W. , Bentele M. , Misteli T. , Rudolf R. , Kaether C. , Spector D. L. , Gerdes H. H. , Eils R.
Published: July 2002

Recent development of
in vivo
microscopy techniques, including green fluorescent proteins, has allowed the visualization of a wide range of dynamic processes in living cells. For quantitative and visual interpretation of such processes, new concepts for time-resolved image analysis and continuous time–space visualization are required. Here, we describe a versatile and fully automated approach consisting of four techniques, namely highly sensitive object detection, fuzzy logic-based dynamic object tracking, computer graphical visualization, and measurement in time–space. Systematic model simulations were performed to evaluate the reliability of the automated object detection and tracking method. To demonstrate potential applications, the method was applied to the analysis of secretory membrane traffic and the functional dynamics of nuclear compartments enriched in pre-mRNA splicing factors.
LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): Binding sites for the chromophore flavin mononucleotide
Authors: Christie John M. , Salomon Michael , Nozue Kazunari , Wada Masamitsu , Briggs Winslow R.
Published: July 2002

Phototropism, the bending response of plant organs to or away from a directional light source, is one of the best studied blue light responses in plants. Although phototropism has been studied for more than a century, recent advances have improved our understanding of the underlying signaling mechanisms involved. The
NPH1
gene of
Arabidopsis thaliana
encodes a blue light-dependent autophosphorylating protein kinase with the properties of a photoreceptor for phototropism. NPH1 apoprotein noncovalently binds FMN to form the holoprotein nph1. The N-terminal region of the protein contains two LOV (light, oxygen, or voltage) domains that share homology with sensor proteins from a diverse group of organisms. These include the bacterial proteins NIFL and AER, both of which bind FAD, and the phy3 photoreceptor from
Adiantium capillus-veneris
. The LOV domain has therefore been proposed to reflect a flavin-binding site, regulating nph1 kinase activity in response to blue light-induced redox changes. Herein we demonstrate that the LOV domains of two nph1 proteins and phy3 bind stoichiometric amounts of FMN when expressed in
Escherichia coli.
The spectral properties of the chromopeptides are similar to the action spectrum for phototropism, implying that the LOV domain binds FMN to function as a light sensor. Thus, our findings support the earlier model that nph1 is a dual-chromophoric flavoprotein photoreceptor regulating phototropic responses in higher plants. We therefore propose the name phototropin to designate the nph1 holoprotein.
Disrupted cortical map and absence of cortical barrels in growth-associated protein (GAP)-43 knockout mice
Authors: Maier Donna L. , Mani Shyamala , Donovan Stacy L. , Soppet Dan , Tessarollo Lino , McCasland James S. , Meiri Karina F.
Published: July 2002

There is strong evidence that growth-associated protein (GAP-43), a protein found only in the nervous system, regulates the response of neurons to axonal guidance signals. However, its role in complex spatial patterning in cerebral cortex has not been explored. We show that mice lacking GAP-43 expression (−/−) fail to establish the ordered whisker representation (barrel array) normally found in layer IV of rodent primary somatosensory cortex. Thalamocortical afferents to −/− cortex form irregular patches in layer IV within a poorly defined cortical field, which varies between hemispheres, rather than the stereotypic, whisker-specific, segregated map seen in normal animals. Furthermore, many thalamocortical afferents project abnormally to widely separated cortical targets. Taken together, our findings indicate a loss of identifiable whisker territories in the GAP-43 −/− mouse cortex. Here, we present a disrupted somatotopic map phenotype in cortex, in clear contrast to the blurring of boundaries within an ordered whisker map in other barrelless mutants. Our results indicate that GAP-43 expression is critical for the normal establishment of ordered topography in barrel cortex.
Studying interactions of four proteins in the yeast two-hybrid system: Structural resemblance of the pVHL/elongin BC/hCUL-2 complex with the ubiquitin ligase complex SKP1/cullin/F-box protein
Authors: Pause Arnim , Peterson Brett , Schaffar Gregor , Stearman Robert , Klausner Richard D.
Published: July 2002

The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners. However, the system fails to detect protein–protein interactions that require the presence of additional components of a multisubunit complex. Here we demonstrate that the vector YIpDCE1 can be used to express elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel–Lindau tumor-suppressor gene product (pVHL). Only when pVHL and elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/elongin BC/hCUL-2 complex. This system was then used to map the binding region of hCUL-2 for the VBC complex. The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex. The elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with elongins B and C. These results reveal a striking structural resemblance of pVHL/elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions. Thus, it seems possible that pVHL/elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome.
Loss of the ataxia–telangiectasia gene product causes oxidative damage in target organs
Authors: Barlow Carrolee , Dennery Phyllis A. , Shigenaga Mark K. , Smith Mark A. , Morrow Jason D. , Roberts L. Jackson , Wynshaw-Boris Anthony , Levine Rodney L.
Published: July 2002

Ataxia–telangiectasia (A-T) is characterized by a markedly increased sensitivity to ionizing radiation, increased incidence of cancer, and neurodegeneration, especially of the cerebellar Purkinje cells. Ionizing radiation oxidizes macromolecules and causes tissue damage through the generation of reactive oxygen species (ROS). We therefore hypothesized that A-T is due to oxidative damage resulting from loss of function of the A-T gene product. To assess this hypothesis, we employed an animal model of A-T, the mouse with a disrupted
Atm
gene. We show that organs which develop pathologic changes in the
Atm
-deficient mice are targets of oxidative damage, and that cerebellar Purkinje cells are particularly affected. These observations provide a mechanistic basis for the A-T phenotype and lay a rational foundation for therapeutic intervention.
Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer β-amyloid peptides
Authors: Greenfield Jeffrey P. , Tsai Julia , Gouras Gunnar K. , Hai Bing , Thinakaran Gopal , Checler Frederic , Sisodia Sangram S. , Greengard Paul , Xu Huaxi
Published: July 2002

The excessive generation and accumulation of 40- and 42-aa β-amyloid peptides (Aβ
40
/Aβ
42
) in selectively vulnerable brain regions is a major neuropathological feature of Alzheimer’s disease. Aβ, derived by proteolytic cleavage from the β-amyloid precursor protein (βAPP), is normally secreted. However, recent evidence suggests that significant levels of Aβ also may remain inside cells. Here, we have investigated the subcellular compartments within which distinct amyloid species are generated and the compartments from which they are secreted. Three experimental approaches were used: (
i
) immunofluorescence performed in intact cortical neurons; (
ii
) sucrose gradient fractionation performed with mouse neuroblastoma cells stably expressing wild-type βAPP
695
(N2a
695
); and (
iii
) cell-free reconstitution of Aβ generation and trafficking from N2a
695
cells. These studies demonstrate that: (
i
) Aβ
40
(Aβ
1–40
plus Aβ
x-40
, where x is an NH
2
-terminal truncation) is generated exclusively within the trans-Golgi Network (TGN) and packaged into post-TGN secretory vesicles; (
ii
) Aβ
x-42
is made and retained within the endoplasmic reticulum in an insoluble state; (
iii
) Aβ
42
(Aβ
1–42
plus Aβ
x-42
) is made in the TGN and packaged into secretory vesicles; and (
iv
) the amyloid peptides formed in the TGN consist of two pools (a soluble population extractable with detergents and a detergent-insoluble form). The identification of the organelles in which distinct forms of Aβ are generated and from which they are secreted should facilitate the identification of the proteolytic enzymes responsible for their formation.