bluuebunny/crossref_metadata_2025 huggingface dataset:
Gap-junctional coupling between neurons and astrocytes in primary central nervous system cultures
Authors: Fróes Maira M. , Correia Ana Helena P. , Garcia-Abreu José , Spray David C. , Campos de Carvalho Antônio C. , Neto Vivaldo Moura
Published: July 2002

Gap-junctional communication between neurons and astrocytes dissociated from rat brain was identified in culture by using dye-transfer assays and electrophysiological measurements. Cell types were identified by using antibodies against β-tubulin III, glial fibrillary acidic protein, and 2′,3′-cyclic-nucleotide phosphohydrolase, which are antigenic determinants of neurons, astroglia, and oligodendrocytes, respectively. Dye coupling was examined as a function of time after dissociated embryonic brain cells were plated onto confluent monolayers of postnatal astrocytes by intracellularly injecting the fluorochrome Lucifer yellow. Coupling of neurons to the astrocytic monolayer was most frequent between 48 h and 72 h in culture and declined over the next 4 days. This gradual uncoupling was accompanied by progressive neuronal maturation, as indicated by morphological measurements in camera lucida drawings. Dye spread was abolished reversibly by octanol, an agent that blocks gap junction channels in other systems. Double whole-cell voltage-clamp measurements confirmed the presence of heterocellular electrical coupling in these cocultures. Coupling was also seen between neurons and astrocytes in cocultures of cells dissociated from embryonic cerebral hemispheres but was rarely detectable in cocultures of postnatal brain cells. These data strongly suggest that junctional communication may provide metabolic and electrotonic interconnections between neuronal and astrocytic networks at early stages of neural development and that such interactions are weakened as differentiation progresses.
Transcriptional and posttranslational plasticity and the generation of inflammatory pain
Authors: Woolf Clifford J. , Costigan Michael
Published: July 2002

Inflammatory pain manifests as spontaneous pain and pain
hypersensitivity. Spontaneous pain reflects direct activation of
specific receptors on nociceptor terminals by inflammatory mediators.
Pain hypersensitivity is the consequence of early posttranslational
changes, both in the peripheral terminals of the nociceptor and in
dorsal horn neurons, as well as later transcription-dependent changes
in effector genes, again in primary sensory and dorsal horn neurons.
This inflammatory neuroplasticity is the consequence of a combination
of activity-dependent changes in the neurons and specific signal
molecules initiating particular signal-transduction pathways. These
pathways phosphorylate membrane proteins, changing their function, and
activate transcription factors, altering gene expression. Two distinct
aspects of sensory neuron function are changed as a result of these
processes, basal sensitivity, or the capacity of peripheral stimuli to
evoke pain, and stimulus-evoked hypersensitivity, the capacity of
certain inputs to generate prolonged alterations in the sensitivity of
the system. Posttranslational changes largely alter basal sensitivity.
Transcriptional changes both potentiate the system and alter neuronal
phenotype. Potentiation occurs as a result of the up-regulation in the
dorsal root ganglion of centrally acting neuromodulators and
simultaneously in the dorsal horn of their receptors. This means that
the response to subsequent inputs is augmented, particularly those that
induce stimulus-induced hypersensitivity. Alterations in phenotype
includes the acquisition by A fibers of neurochemical features typical
of C fibers, enabling these fibers to induce stimulus-evoked
hypersensitivity, something only C fiber inputs normally can do.
Elucidation of the molecular mechanisms responsible provides new
opportunities for therapeutic approaches to managing inflammatory pain.
Apoptosis in heart failure: Release of cytochrome
c
from mitochondria and activation of caspase-3 in human cardiomyopathy
Authors: Narula Jagat , Pandey Pramod , Arbustini Eloisa , Haider Nezam , Narula Navneet , Kolodgie Frank D. , Dal Bello Barbara , Semigran Marc J. , Bielsa-Masdeu Anna , Dec G. William , Israels Sara , Ballester Manel , Virmani Renu , Saxena Satya , Kharbanda Surender
Published: July 2002

Apoptosis has been shown to contribute to loss of cardiomyocytes in cardiomyopathy, progressive decline in left ventricular function, and congestive heart failure. Because the molecular mechanisms involved in apoptosis of cardiocytes are not completely understood, we studied the biochemical and ultrastructural characteristics of upstream regulators of apoptosis in hearts explanted from patients undergoing transplantation. Sixteen explanted hearts from patients undergoing heart transplantation were studied by electron microscopy or immunoblotting to detect release of mitochondrial cytochrome
c
and activation of caspase-3. The hearts explanted from five victims of motor vehicle accidents or myocardial ventricular tissues from three donor hearts were used as controls. Evidence of apoptosis was observed only in endstage cardiomyopathy. There was significant accumulation of cytochrome
c
in the cytosol, over myofibrils, and near intercalated discs of cardiomyocytes in failing hearts. The release of mitochondrial cytochrome
c
was associated with activation of caspase-3 and cleavage of its substrate protein kinase C δ but not poly(ADP-ribose) polymerase. By contrast, there was no apparent accumulation of cytosolic cytochrome
c
or caspase-3 activation in the hearts used as controls. The present study provides
in vivo
evidence of cytochrome
c
-dependent activation of cysteine proteases in human cardiomyopathy. Activation of proteases supports the phenomenon of apoptosis in myopathic process. Because loss of myocytes contributes to myocardial dysfunction and is a predictor of adverse outcomes in the patients with congestive heart failure, the present demonstration of an activated apoptotic cascade in cardiomyopathy could provide the basis for novel interventional strategies.
Ultrafast detection and control of molecular dynamics
Authors: Anfinrud Philip , de Vivie-Riedle Regina , Engel Volker
Published: July 2002

Many elementary chemical and physical processes such as the
breaking of a chemical bond or the vibrational motion of atoms within a
molecule take place on a femtosecond (fs = 10
−15
s)
or picosecond (ps = 10
−12
s) time scale. It is now
possible to monitor these events as a function of time with temporal
resolution well below 100 fs. This capability is based on the
pump-probe technique where one optical pulse triggers a reaction and a
second delayed optical pulse probes the changes that ensue. To
illustrate this capability, the dynamics of ligand motion within a
protein are presented. Moving beyond casual observation of a reaction
to active control of its outcome requires additional experimental and
theoretical effort. To illustrate the concept of control, the effect of
optical pulse duration on the vibrational dynamics of a tri-atomic
molecule are discussed. The experimental and theoretical resources
currently available are poised to make the dream of reaction control a
reality for certain molecular systems.
Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction
Authors: Li Cai , Friedman Jeffrey M.
Published: July 2002

Leptin exerts its weight-reducing effects by binding to its receptor and activating signal transduction in hypothalamic neurons and other cell types. To identify the components of the leptin signal transduction pathway, an approach was developed in which bacterially expressed phosphorylated fragments of Ob receptor b (Ob-Rb) were used as affinity agents. Leptin binding to the Ob-Rb form of the leptin receptor leads to tyrosyl phosphorylation of the cytoplasmic domain of its receptor. Two of the three cytoplasmic tyrosines of Ob-Rb, at positions 985 and 1138, are phosphorylated after leptin treatment. Affinity chromatography using a tyrosine-phosphorylated fragment spanning Tyr 985 of Ob-Rb was used to identify proteins that bind to this site. The SH2 domain containing protein tyrosine phosphatase 2 (SHP-2) was isolated from bovine and mouse hypothalamus by using this method. After cotransfection of Ob-Rb, Janus kinase 2 (JAK2), and SHP-2 into 293T cells, leptin results in direct binding of SHP-2 to the phosphorylated Tyr 985. The bound SHP-2 is itself tyrosine phosphorylated after leptin treatment. SHP-2 is not phosphorylated after leptin treatment when a Y→F 985 receptor mutant is cotransfected. In the absence of SHP-2 phosphorylation, the level of JAK2 phosphorylation was increased. Tyrosyl phosphorylation of the leptin receptor and signal transducer and activater of transcription 3 (STAT3) are not affected by phosphorylation of SHP-2. These data suggest that activation of SHP-2 by the leptin receptor results in a decreased phosphorylation of JAK2 and may act to attenuate leptin signal transduction. The method used in this report can in principle be used to isolate additional components of the leptin, or other, signal transduction pathway.
Cytoplasmic amino and carboxyl domains form a wide intracellular vestibule in an inwardly rectifying potassium channel
Authors: Lu Tao , Zhu Yong-gang , Yang Jian
Published: July 2002

We have studied the structural components and architecture of the intracellular vestibule of a strongly rectifying channel (Kir2.1) expressed in
Xenopus
oocytes. Putative vestibule-lining residues were identified by systematically examining covalent modification by sulfhydryl-specific reagents of cysteine residues engineered into two cytoplasmic regions. In a stretch of 33 amino acids in the amino terminus (from C54 to V86) and 22 amino acids in the carboxyl terminus (from R213 to S234), 15 and 11 residues, respectively, were found to be accessible to methanethiosulfonate ethylammonium (MTSEA) or methanethiosulfonate ethyltrimethylammonium (MTSET) and presumably project into the aqueous intracellular vestibule. The pattern of accessibility suggests that both stretches may adopt an extended loop structure. To explore the physical dimension of the intracellular vestibule, we covalently linked a constrained number (one to four) of positively charged moieties of different sizes to the E224 position and found that this vestibule region is sufficiently wide to accommodate four modifying groups with dimensions of 12 Å × 10 Å × 6 Å. These results suggest that regions in both the amino and carboxyl domains of Kir2.1 channel form a long and wide intracellular vestibule that protrudes beyond the membrane into the cytoplasm.
Attenuated sensitivity to neuroactive steroids in γ-aminobutyrate type A receptor delta subunit knockout mice
Authors: Mihalek Robert M. , Banerjee Pradeep K. , Korpi Esa R. , Quinlan Joseph J. , Firestone Leonard L. , Mi Zhi-Ping , Lagenaur Carl , Tretter Verena , Sieghart Werner , Anagnostaras Stephan G. , Sage Jennifer R. , Fanselow Michael S. , Guidotti Alessandro , Spigelman Igor , Li Zhiwei , DeLorey Timothy M. , Olsen Richard W. , Homanics Gregg E.
Published: July 2002

γ-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the δ subunit of the GABA type A receptors.
In vivo
testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the δ subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.
Low-status monkeys “play dumb” when learning in mixed social groups
Authors: Drea Christine M. , Wallen Kim
Published: July 2002

Many primates, including humans, live in complex hierarchical societies where social context and status affect daily life. Nevertheless, primate learning studies typically test single animals in limited laboratory settings where the important effects of social interactions and relationships cannot be studied. To investigate the impact of sociality on associative learning, we compared the individual performances of group-tested rhesus monkeys (
Macaca mulatta
) across various social contexts. We used a traditional discrimination paradigm that measures an animal’s ability to form associations between cues and the obtaining of food in choice situations; but we adapted the task for group testing. After training a 55-member colony to separate on command into two subgroups, composed of either high- or low-status families, we exposed animals to two color discrimination problems, one with all monkeys present (combined condition), the other in their “dominant” and “subordinate” cohorts (split condition). Next, we manipulated learning history by testing animals on the same problems, but with the social contexts reversed. Monkeys from dominant families excelled in all conditions, but subordinates performed well in the split condition only, regardless of learning history. Subordinate animals had learned the associations, but expressed their knowledge only when segregated from higher-ranking animals. Because aggressive behavior was rare, performance deficits probably reflected voluntary inhibition. This experimental evidence of rank-related, social modulation of performance calls for greater consideration of social factors when assessing learning and may also have relevance for the evaluation of human scholastic achievement.
Measurement of community metabolism and significance in the coral reef CO
2
source-sink debate
Authors: Gattuso Jean-Pierre , Frankignoulle Michel , Smith Stephen V.
Published: July 2002

Two methods are commonly used to measure the community metabolism (primary production, respiration, and calcification) of shallow-water marine communities and infer air–sea CO
2
fluxes: the pH-total alkalinity and pH-O
2
techniques. The underlying assumptions of each technique are examined to assess the recent claim that the most widely used technique in coral reefs (pH-total alkalinity), may have provided spurious results in the past because of high rates of nitrification and release of phosphoric acid in the water column [Chisholm, J. R. M. & Barnes, D. J. (1998)
Proc. Natl. Acad. Sci. USA
95, 6566–6569]. At least three lines of evidence suggest that this claim is not founded. First, the rate of nitrification required to explain the discrepancy between the two methods recently reported is not realistic as it is much higher than the rates measured in another reef system and greater than the highest rate measured in a marine environment. Second, fluxes of ammonium, nitrate, and phosphorus are not consistent with high rates of nitrification and release of phosphoric acid. Third, the consistency of the metabolic parameters obtained by using the two techniques is in good agreement in two sites recently investigated. The pH-total alkalinity technique therefore appears to be applicable in most coral reef systems. Consequently, the conclusion that most coral reef flats are sources of CO
2
to the atmosphere does not need revision. Furthermore, we provide geochemical evidence that calcification in coral reefs, as well as in other calcifying ecosystems, is a long-term source of CO
2
for the atmosphere.
Regulation of the dolichol pathway in human fibroblasts by the endoplasmic reticulum unfolded protein response
Authors: Doerrler William T. , Lehrman Mark A.
Published: July 2002

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) of eukaryotic cells triggers the unfolded protein response (UPR), which activates transcription of several genes encoding ER chaperones and folding enzymes. This study reports that conversion of dolichol-linked Man
2–5
GlcNAc
2
intermediates into mature Glc
3
Man
9
GlcNAc
2
oligosaccharides in primary human adult dermal fibroblasts is also stimulated by the UPR. This stimulation was not evident in several immortal cell lines and did not require a cytoplasmic stress response. Inhibition of dolichol-linked Glc
3
Man
9
GlcNAc
2
synthesis by glucose deprivation could be counteracted by the UPR, improving the transfer of Glc
3
Man
9
GlcNAc
2
to asparagine residues on nascent polypeptides. Glycosidic processing of asparagine-linked Glc
3
Man
9
GlcNAc
2
in the ER leads to the production of monoglucosylated oligosaccharides that promote interaction with the lectin chaperones calreticulin and calnexin. Thus, control of the dolichol-linked Glc
3
Man
9
GlcNAc
2
supply gives the UPR the potential to maintain efficient protein folding in the ER without new synthesis of chaperones or folding enzymes.
Engagement of p75/AIRM1 or CD33 inhibits the proliferation of normal or leukemic myeloid cells
Authors: Vitale Chiara , Romagnani Chiara , Falco Michela , Ponte Marco , Vitale Massimo , Moretta Alessandro , Bacigalupo Andrea , Moretta Lorenzo , Mingari Maria Cristina
Published: July 2002

P75/AIRM1 is a recently identified surface molecule that belongs to the sialoadhesin family and displays homology with the myeloid cell antigen CD33. In lymphoid cells, p75/AIRM1 is confined to natural killer cells and mediates inhibition of their cytolytic activity. In this study, we show that p75/AIRM1 is also expressed by cells of the myelomonocytic cell lineage, in which it appears at a later stage as compared with CD33.
In vitro
proliferation and differentiation of cord blood-derived CD34
+
cells (induced by stem cell factor and granulocyte–macrophage colony-stimulating factor) were consistently inhibited by the addition of anti-p75/AIRM1 mAb. Engagement of CD33 led to inhibition in some experiments. A sharp decrease of cell proliferation/survival was detected in all three p75/AIRM1+ chronic myeloid leukemias analyzed when cultured in the presence of either anti-p75/AIRM1 or anti-CD33 mAbs. Thus, the present study suggests that p75/AIRM1 and CD33 may play a regulatory role in normal myelopoiesis and may be viewed as suitable target molecules to counteract the proliferation/survival of chronic myeloid leukemias.
Neuronal calcium sparks and intracellular calcium “noise”
Authors: Melamed-Book Naomi , Kachalsky Sylvia G. , Kaiserman Igor , Rahamimoff Rami
Published: July 2002

Intracellular calcium ions are involved in many forms of cellular function. To accommodate so many control functions, a complex spatiotemporal organization of calcium signaling has developed. In both excitable and nonexcitable cells, calcium signaling was found to fluctuate. Sudden localized increases in the intracellular calcium concentration—or calcium sparks—were found in heart, striated and smooth muscle,
Xenopus
Laevis
oocytes, and HeLa and P12 cells. In the nervous system, intracellular calcium ions were found important in key processes such as transmitter release, repetitive firing, and gene expression. Hence, we examined whether calcium sparks also exist in neurons. Using confocal laser-scanning microscopy and fluorescent probes, we found that calcium sparks exist in two types of neuronal preparations: the presynaptic boutons of the lizard neuromuscular junction and rat hippocampal neurons in cell culture. Control experiments exclude the possibility that these calcium sparks originate from instrumental or biological artifacts. Calcium sparks seem to be just the tip of the iceberg of a more general phenomenon of intracellular calcium “noise.” We speculate that calcium sparks and calcium noise may be of key importance in calcium signaling in the nervous system.
Model of maltose-binding protein/chemoreceptor complex supports intrasubunit signaling mechanism
Authors: Zhang Yinghua , Gardina Paul J. , Kuebler Ann S. , Kang Hui S. , Christopher Jon A. , Manson Michael D.
Published: July 2002

The Tar protein of
Escherichia coli
is unique among known bacterial chemoreceptors in that it generates additive responses to two very disparate ligands, aspartate and maltose. Aspartate binds directly to the periplasmic (extracytoplasmic) domain of Tar. Maltose first binds to maltose-binding protein (MBP). MBP then assumes a closed conformation in which it can interact with the periplasmic domain of Tar. MBP residues critical for binding Tar were identified in a screen of mutations that cause specific defects in maltose chemotaxis. Mutations were introduced into a plasmid-borne
malE
gene that encodes a mutant form of MBP in which two engineered Cys residues spontaneously generate a disulfide bond in the oxidizing environment of the periplasmic space. This disulfide covalently crosslinks the NH
3
-terminal and COOH-terminal domains of MBP and locks the protein into a closed conformation. Double-Cys MBP confers a dominant-negative phenotype for maltose taxis, and we reasoned that third mutations that relieve this negative dominance probably alter residues that are important for the initial interaction of MBP with Tar. The published three-dimensional structures of MBP and the periplasmic domain of
E. coli
Tar were docked in a computer simulation that juxtaposed the residues in MBP identified in this way with residues in Tar that have been implicated in maltose taxis. The resulting model of the MBP-Tar complex exhibits good complementarity between the surfaces of the two proteins and supports the idea that aspartate and MBP may each initiate an attractant signal through Tar by inducing similar conformational changes in the chemoreceptor.
Blockade of type β transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat
Authors: Qi Zhe , Atsuchi Nobuhiko , Ooshima Akira , Takeshita Akira , Ueno Hikaru
Published: July 2002

We eliminated type β transforming growth factor (TGF-β) signaling by adenovirus-mediated local expression of a dominant-negative type II TGF-β receptor (AdCATβ-TR) in the liver of rats treated with dimethylnitrosamine, a model of persistent liver fibrosis. In rats that received a single application of AdCATβ-TR via the portal vein, liver fibrosis as assessed by histology and hydroxyproline content was markedly attenuated. All AdCATβ-TR-treated rats remained alive, and their serum levels of hyaluronic acid and transaminases remained at low levels, whereas all the AdCATβ-TR-untreated rats died of liver dysfunction. The results demonstrate that TGF-β does play a central role in liver fibrogenesis and indicate clearly in a persistent fibrosis model that prevention of fibrosis by anti-TGF-β intervention could be therapeutically useful.
Transcriptional activation by artificial recruitment in yeast is influenced by promoter architecture and downstream sequences
Authors: Gaudreau Luc , Keaveney Marie , Nevado Julián , Zaman Zafar , Bryant Gene O. , Struhl Kevin , Ptashne Mark
Published: July 2002

The idea that recruitment of the transcriptional machinery to a promoter suffices for gene activation is based partly on the results of “artificial recruitment” experiments performed
in vivo
. Artificial recruitment can be effected by a “nonclassical” activator comprising a DNA-binding domain fused to a component of the transcriptional machinery. Here we show that activation by artificial recruitment in yeast can be sensitive to any of three factors: position of the activator-binding elements, sequence of the promoter, and coding sequences downstream of the promoter. In contrast, classical activators worked efficiently at all promoters tested. In all cases the “artificial recruitment” fusions synergized well with classical activators. A classical activator evidently differs from a nonclassical activator in that the former can touch multiple sites on the transcriptional machinery, and we propose that that difference accounts for the broader spectrum of activity of the typical classical activator. A similar conclusion is reached from studies in mammalian cells in the accompanying paper [Nevado, J., Gaudreau, L., Adam, M. & Ptashne, M. (1999)
Proc. Natl. Acad. Sci. USA
96, 2674–2677].
Nitroreductase A is regulated as a member of the
soxRS
regulon of
Escherichia coli
Authors: Liochev Stefan I. , Hausladen Alfred , Fridovich Irwin
Published: July 2002

Nitroreductase A catalyzes the divalent reduction of nitro compounds, quinones, and dyes by NADPH. In this paper, nitroreductase A is induced in
Escherichia coli
by exposure to paraquat in a manner that depends on the expression of
soxR
. Nitroreductase activity was only slightly induced by paraquat in a strain bearing a mutational defect in the gene encoding nitroreductase A, but it was ≈3-fold induced in the parental strain. Nitroreductase A thus appears to be a member of the
soxRS
regulon and probably contributes to the defenses against oxidative stress by minimizing the redox cycling attendant upon the univalent reduction of nitro compounds, quinones, and dyes.
Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Förster distance dependence and subpopulations
Authors: Deniz Ashok A. , Dahan Maxime , Grunwell Jocelyn R. , Ha Taekjip , Faulhaber Ann E. , Chemla Daniel S. , Weiss Shimon , Schultz Peter G.
Published: July 2002

Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.
Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea
Authors: Haqqi Tariq M. , Anthony Donald D. , Gupta Sanjay , Ahmad Nihal , Lee M.-S. , Kumar Ganesh K. , Mukhtar Hasan
Published: July 2002

Identification of common dietary substances capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. An antioxidant-rich polyphenolic fraction isolated from green tea (green tea polyphenols, GTPs) has been shown to possess anti-inflammatory and anticarcinogenic properties in experimental animals. In this study we determined the effect of oral consumption of GTP on collagen-induced arthritis in mice. In three independent experiments mice given GTP in water exhibited significantly reduced incidence of arthritis (33% to 50%) as compared with mice not given GTP in water (84% to 100%). The arthritis index also was significantly lower in GTP-fed animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-γ, and tumor necrosis factor α in arthritic joints of GTP-fed mice. Histologic and immunohistochemical analysis of the arthritic joints in GTP-fed mice demonstrated only marginal joint infiltration by IFN-γ and tumor necrosis factor α-producing cells as opposed to massive cellular infiltration and fully developed pannus in arthritic joints of non-GTP-fed mice. The neutral endopeptidase activity was approximately 7-fold higher in arthritic joints of non-GTP-fed mice in comparison to nonarthritic joints of unimmunized mice whereas it was only 2-fold higher in the arthritic joints of GTP-fed mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of GTP-fed mice. Taken together our studies suggest that a polyphenolic fraction from green tea that is rich in antioxidants may be useful in the prevention of onset and severity of arthritis.
Mitochondrial disease in mouse results in increased oxidative stress
Authors: Esposito Luke A. , Melov Simon , Panov Alexander , Cottrell Barbara A. , Wallace Douglas C.
Published: July 2002

It has been hypothesized that a major factor in the progression of mitochondrial disease resulting from defects in oxidative phosphorylation (OXPHOS) is the stimulation of the mitochondrial production of reactive oxygen species (ROS) and the resulting damage to the mtDNA. To test this hypothesis, we examined the mitochondria from mice lacking the heart/muscle isoform of the adenine nucleotide translocator (
Ant1
), designated
Ant1
tm2Mgr
(−/−) mice. The absence of
Ant1
blocks the exchange of ADP and ATP across the mitochondrial inner membrane, thus inhibiting OXPHOS. Consistent with
Ant1
expression, mitochondria isolated from skeletal muscle, heart, and brain of the
Ant1
-deficient mice produced markedly increased amounts of the ROS hydrogen peroxide, whereas liver mitochondria, which express a different
Ant
isoform, produced normally low levels of hydrogen peroxide. The increased production of ROS by the skeletal muscle and heart was associated with a dramatic increase in the ROS detoxification enzyme manganese superoxide dismutase (Sod2, also known as MnSod) in muscle tissue and muscle mitochondria, a modest increase in Sod2 in heart tissue, and no increase in heart mitochondria. The level of glutathione peroxidase-1 (Gpx1), a second ROS detoxifying enzyme, was increased moderately in the mitochondria of both tissues. Consistent with the lower antioxidant defenses in heart, the heart mtDNAs of the
Ant1
-deficient mice showed a striking increase in the accumulation of mtDNA rearrangements, whereas skeletal muscle, with higher antioxidant defenses, had fewer mtDNA rearrangements. Hence, inhibition of OXPHOS does increase mitochondrial ROS production, eliciting antioxidant defenses. If the antioxidant defenses are insufficient to detoxify the ROS, then an increased mtDNA mutation rate can result.
huASH1 protein, a putative transcription factor encoded by a human homologue of the
Drosophila ash1
gene, localizes to both nuclei and cell–cell tight junctions
Authors: Nakamura Tatsuya , Blechman Janna , Tada Shinichiro , Rozovskaia Tanya , Itoyama Takahiro , Bullrich Florencia , Mazo Alexander , Croce Carlo M. , Geiger Benjamin , Canaani Eli
Published: July 2002

During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox/homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the
trithorax
(trx) and
Polycomb
(
Pc
) groups of genes
. trithorax (trx)
and
ash1
(absent, small, or homeotic 1) are members of the
Drosophila trx
group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Recently, we reported molecular studies indicating that TRX and ASH1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of
ash1
(the mammalian homologue of
trx
, ALL-1, was previously cloned). We have identified a human expressed sequence tag (EST) clone with similarity to the SET domain of
Drosophila
ASH1, and used it to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an ≈10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in
Drosophila
ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, we found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. Double-immunofluorescence labeling of huASH1 and several junctional proteins localized the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, we consider the possibility that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesion-mediated signaling.
Crystal structure of human stem cell factor: Implication for stem cell factor receptor dimerization and activation
Authors: Zhang Zhongtao , Zhang Rongguang , Joachimiak Andrzej , Schlessinger Joseph , Kong Xiang-Peng
Published: July 2002

Stem cell factor (SCF) plays important roles in hematopoiesis and the survival, proliferation, and differentiation of mast cells, melanocytes, and germ cells. SCF mediates its biological effects by binding to and activating a receptor tyrosine kinase designated c-kit or SCF receptor. In this report we describe the 2.3-Å crystal structure of the functional core of recombinant human SCF. SCF is a noncovalent homodimer composed of two slightly wedged protomers. Each SCF protomer exhibits an antiparallel four-helix bundle fold. Dimerization is mediated by extensive polar and nonpolar interactions between the two protomers with a large buried surface area. Finally, we have identified a hydrophobic crevice and a charged region at the tail of each protomer that functions as a potential receptor-binding site. On the basis of these observations, a model for SCF⋅c-kit complex formation and dimerization is proposed.
Overexpression of angiotensin II type I receptor in cardiomyocytes induces cardiac hypertrophy and remodeling
Authors: Paradis Pierre , Dali-Youcef Nassim , Paradis François W. , Thibault Gaétan , Nemer Mona
Published: July 2002

Angiotensin II (AII) is a major determinant of arterial pressure and volume homeostasis, mainly because of its vascular action via the AII type 1 receptor (AT1R). AII has also been implicated in the development of cardiac hypertrophy because angiotensin I-converting enzyme inhibitors and AT1R antagonists prevent or regress ventricular hypertrophy in animal models and in human. However, because these treatments impede the action of AII at cardiac as well as vascular levels, and reduce blood pressure, it has been difficult to determine whether AII action on the heart is direct or a consequence of pressure-overload. To determine whether AII can induce cardiac hypertrophy directly via myocardial AT1R in the absence of vascular changes, transgenic mice overexpressing the human AT1R under the control of the mouse α-myosin heavy chain promoter were generated. Cardiomyocyte-specific overexpression of AT1R induced, in basal conditions, morphologic changes of myocytes and nonmyocytes that mimic those observed during the development of cardiac hypertrophy in human and in other mammals. These mice displayed significant cardiac hypertrophy and remodeling with increased expression of ventricular atrial natriuretic factor and interstitial collagen deposition and died prematurely of heart failure. Neither the systolic blood pressure nor the heart rate were changed. The data demonstrate a direct myocardial role for AII in the development of cardiac hypertrophy and failure and provide a useful model to elucidate the mechanisms of action of AII in the pathogenesis of cardiac diseases.
Direct genetic demonstration of Gα13 coupling to the orphan G protein-coupled receptor G2A leading to RhoA-dependent actin rearrangement
Authors: Kabarowski Janusz H. S. , Feramisco Jamison D. , Le Lu Q. , Gu Jennifer L. , Luoh Shiuh-Wen , Simon Melvin I. , Witte Owen N.
Published: July 2002

G2A is an orphan G protein-coupled receptor (GPCR), expressed predominantly in T and B cells and homologous to a small group of GPCRs of unknown function expressed in lymphoid tissues. G2A is transcriptionally induced in response to diverse stimuli, and its ectopic expression suppresses transformation of B lymphoid precursors by BCR-ABL. G2A induces morphological transformation of NIH 3T3 fibroblasts. Microinjection of constructs encoding G2A into Swiss 3T3 fibroblasts induces actin reorganization into stress fibers that depends on RhoA, but not CDC42 or RAC. G2A elicits RhoA-dependent transcriptional activation of serum response factor. Direct evaluation of RhoA activity demonstrates elevated levels of RhoA-GTP in G2A-expressing cells. Microinjection of embryonic fibroblasts derived from various Gα knockout mice establishes a requirement for Gα13 but not Gα12 or Gαq/11 in G2A-induced actin rearrangement. In conclusion, G2A represents a family of GPCRs expressed in lymphocytes that may link diverse stimuli to cytoskeletal reorganization and transcriptional activation through a pathway involving Gα13 and RhoA.
Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation
Authors: Du Xue-Liang , Edelstein Diane , Rossetti Luciano , Fantus Ivan George , Goldberg Howard , Ziyadeh Fuad , Wu Jie , Brownlee Michael
Published: July 2002

The hexosamine pathway has been implicated in the pathogenesis of
diabetic complications. We determined first that hyperglycemia induced
a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in
bovine aortic endothelial cells via increased production of
mitochondrial superoxide and a concomitant 2.4-fold increase in
hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate
dehydrogenase activity and increased hexosamine pathway activity were
prevented completely by an inhibitor of electron transport complex II
(thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation
(carbonyl cyanide
m
-chlorophenylhydrazone), a
superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid)
porphyrin], overexpression of either uncoupling protein 1 or manganese
superoxide dismutase, and azaserine, an inhibitor of the rate-limiting
enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate
amidotransferase). Immunoprecipitation of Sp1 followed by Western
blotting with antibodies to
O-
linked GlcNAc,
phosphoserine, and phosphothreonine showed that hyperglycemia increased
GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased
phosphothreonine by 70%. The same inhibitors prevented all these
changes. Hyperglycemia increased expression from a transforming growth
factor-β
1
promoter luciferase reporter by 2-fold and
increased expression from a (−740 to +44) plasminogen activator
inhibitor-1 promoter luciferase reporter gene by nearly 3-fold.
Inhibition of mitochondrial superoxide production or the glucosamine
pathway prevented all these changes. Hyperglycemia increased expression
from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1)
promoter luciferase reporter containing two Sp1 sites in a similar
fashion (3.8-fold). In contrast, hyperglycemia had no effect when the
two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial
superoxide overproduction increases hexosamine synthesis and
O-
glycosylation of Sp1, which activates expression of
genes that contribute to the pathogenesis of diabetic
complications.
The proton to electron stoichiometry of steady-state photosynthesis in living plants: A proton-pumping Q cycle is continuously engaged
Authors: Sacksteder Colette A. , Kanazawa Atsuko , Jacoby Michael E. , Kramer David M.
Published: July 2002

A noninvasive technique is introduced with which relative proton to
electron stoichiometries (H
+
/e
−
ratios)
for photosynthetic electron transfer can be obtained from leaves of
living plants under steady-state illumination. Both electron and proton
transfer fluxes were estimated by a modification of our previously
reported dark-interval relaxation kinetics (DIRK) analysis, in which
processes that occur upon rapid shuttering of the actinic light are
analyzed. Rates of turnover of linear electron transfer through the
cytochrome (cyt)
b
6
f
complex
were estimated by measuring the DIRK signals associated with reduction
of cyt
f
and P
700
. The rates of
proton pumping through the electron transfer chain and the
CF
O
-CF
1
ATP synthase (ATPase) were estimated by
measuring the DIRK signals associated with the electrochromic shifting
of pigments in the light-harvesting complexes. Electron transfer fluxes
were also estimated by analysis of saturation pulse-induced changes in
chlorophyll
a
fluorescence yield. It was shown that the
H
+
/e
−
ratio, with respect to both cyt
b
6
f
complex and photosystem
(PS) II turnover, was constant under low to saturating illumination in
intact tobacco leaves. Because a H
+
/e
−
ratio of 3 at a low light is generally accepted, we infer that this
ratio is maintained under conditions of normal (unstressed)
photosynthesis, implying a continuously engaged, proton-pumping Q cycle
at the cyt
b
6
f
complex.
Bacterial RNA polymerase subunit ω and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly
Authors: Minakhin Leonid , Bhagat Sechal , Brunning Adrian , Campbell Elizabeth A. , Darst Seth A. , Ebright Richard H. , Severinov Konstantin
Published: July 2002

Bacterial DNA-dependent RNA polymerase (RNAP) has subunit
composition β′βα
I
α
II
ω. The role of
ω has been unclear. We show that ω is homologous in sequence and
structure to RPB6, an essential subunit shared in eukaryotic RNAP I,
II, and III. In
Escherichia coli
, overproduction of ω
suppresses the assembly defect caused by substitution of residue 1362
of the largest subunit of RNAP, β′. In yeast, overproduction of RPB6
suppresses the assembly defect caused by the equivalent substitution in
the largest subunit of RNAP II, RPB1. High-resolution structural
analysis of the ω–β′ interface in bacterial RNAP, and comparison
with the RPB6–RPB1 interface in yeast RNAP II, confirms the structural
relationship and suggests a “latching” mechanism for the role of
ω and RPB6 in promoting RNAP assembly.
An astroglia-linked dopamine D2-receptor action in prefrontal cortex
Authors: Khan Zafar U. , Koulen Peter , Rubinstein Marcelo , Grandy David K. , Goldman-Rakic Patricia S.
Published: July 2002

Typical neuroleptic drugs elicit their antipsychotic effects
mainly by acting as antagonists at dopamine D2 receptors. Much of this
activity is thought to occur in the cerebral cortex, where D2 receptors
are found largely in inhibitory GABAergic neurons. Here we confirm this
localization at the electron microscopic level, but additionally show
that a subset of cortical interneurons with low or undetectable
expression of D2 receptor isoforms are surrounded by astrocytic
processes that strongly express D2 receptors. Ligand binding of
isolated astrocyte preparations indicate that cortical astroglia
account for approximately one-third of the total D2 receptor binding
sites in the cortex, a proportion that we found conserved among rodent,
monkey, and human tissues. Further, we show that the D2
receptor-specific agonist, quinpirole, can induce Ca
2+
elevation in isolated cortical astrocytes in a pharmacologically
reversible manner, thus implicating this receptor in the signaling
mechanisms by which astrocytes communicate with each other as well as
with neurons. The discovery of D2 receptors in astrocytes with a
selective anatomical relationship to interneurons represents a
neuron/glia substrate for cortical dopamine action in the adult
cerebral cortex and a previously unrecognized site of action for
antipsychotic drugs with affinities at the D2 receptor.
Going for Growth: Prestige Projects in Three British Cities
Authors: Loftman Patrick , Nevin Brendan
Published: July 2002

In response to the global restructuring of industry in the 1970s and 1980s, the elected governments of economically depressed cities have increasingly adopted growth-orientated local economic development policies. A key component of pro-growth local economic development strategies, adopted by UK city governments and central government agencies has been investment in, and promotion of, high-profile prestige property developments and civic boosterism activities. This paper provides a review of the local economic development responses of three UK local authorities (Birmingham, Sheffield and Manchester) to economic restructuring and the intensification of inter-city competition. Through an examination of the three case studies, the paper highlights: the debate concerning the validity of pro-growth, local economic development policies; the rationales behind the adoption of such policies; the financing and implementation of pro-growth strategies; economic impacts; and the distributional consequences of pro-growth strategies focused on prestige developments.
Job Proximity and the Urban Employment Problem: Do Suitable Nearby Jobs Improve Neighbourhood Employment Rates?
Authors: Immergluck Daniel
Published: July 2002

Due to a scarcity of small-area jobs data, much of the spatial mismatch literature has not directly addressed the impact of nearby jobs on neighbourhood employment rates. Such analysis is particularly needed when considering the probable effects of neighbourhood-targeted economic development. Moreover, the occupational mix of jobs and their match with resident skills have not been dealt with adequately. A consistent measure of job proximity is found to have a significant but modest effect on neighbourhood employment and unemployment rates, with a standard deviation increase resulting in an increase in the employment rate of approximately six-tenths of a percentage point and a reduction in unemployment of approximately three-tenths of a percentage point. When considering occupational match and the average occupational level of nearby jobs, the effect of nearby jobs is larger. Race and educational attainment are found to have the largest effects on employment rates.
Using Projective Techniques in Education Research
Authors: Catterall Miriam , Ibbotson Patrick
Published: July 2002

Projective techniques are unusual and often intriguing for respondents to complete, permitting them to express thoughts and feelings which can be difficult to access by direct and structured questioning. This is achieved by presenting respondents with ambiguous verbal or visual stimulus materials, such as bubble cartoons, which they need to make sense of by drawing from their own experiences, thoughts, feelings and imagination before they can offer a response. Importantly, projective techniques can be fun and engaging for respondents, especially when they become involved in their analysis and interpretation. The various types of projective techniques are described and their benefits and drawbacks examined. A project involving students completing a range of projective techniques is used to illustrate these benefits and drawbacks.