bluuebunny/crossref_metadata_2025 huggingface dataset:
A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding
Authors: Krylov Dmitry , Kasai Kenji , Echlin Deborah R. , Taparowsky Elizabeth J. , Arnheiter Heinz , Vinson Charles
Published: July 2002

We describe a method to design dominant-negative proteins (D-N) to the basic helix–loop–helix–leucine zipper (B-HLHZip) family of sequence-specific DNA binding transcription factors. The D-Ns specifically heterodimerize with the B-HLHZip dimerization domain of the transcription factors and abolish DNA binding in an equimolar competition. Thermal denaturation studies indicate that a heterodimer between a Myc B-HLHZip domain and a D-N consisting of a 12-amino acid sequence appended onto the Max dimerization domain (A-Max) is −6.3 kcal⋅mol
−1
more stable than the Myc:Max heterodimer. One molar equivalent of A-Max can totally abolish the DNA binding activity of a Myc:Max heterodimer. This acidic extension also has been appended onto the dimerization domain of the B-HLHZip protein Mitf, a member of the transcription factor enhancer binding subfamily, to produce A-Mitf. The heterodimer between A-Mitf and the B-HLHZip domain of Mitf is −3.7 kcal⋅mol
−1
more stable than the Mitf homodimer. Cell culture studies show that A-Mitf can inhibit Mitf-dependent transactivation both in acidic extension and in a dimerization-dependent manner. A-Max can inhibit Myc-dependent foci formation twice as well as the Max dimerization domain (HLHZip). This strategy of producing D-Ns may be applicable to other B-HLHZip or B-HLH proteins because it provides a method to inhibit the DNA binding of these transcription factors in a dimerization-specific manner.
TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines
Authors: Yang Xinzhen , Gold Moses O. , Tang Derek Ng , Lewis Dorothy E. , Aguilar-Cordova Estuardo , Rice Andrew P. , Herrmann Christine H.
Published: July 2002

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase IIin vitroin a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995)J. Virol. 69, 1612–1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.
Immunohistochemistry of HLA-H, the protein defective in patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract
Authors: Parkkila Seppo , Waheed Abdul , Britton Robert S. , Feder John N. , Tsuchihashi Zenta , Schatzman Randall C. , Bacon Bruce R. , Sly William S.
Published: July 2002

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism that leads to excessive iron storage in the liver and other organs. Recently, between 83 and 100% of HH patients have been found to be homozygous for the same mutation in a novel major histocompatibility complex class I-like gene, called the HLA-H gene. The Cys-282 → Tyr mutation in HH patients would be expected to disrupt the function of the HLA-H gene product by altering a critical disulfide bridge. As a first step in understanding the function of the HLA-H gene product, we generated an antibody to a C-terminal peptide and used it for immunolocalization of the HLA-H protein in the gastrointestinal tract of Finnish and American subjects presumed not to have HH. Although staining for the HLA-H protein was seen in some epithelial cells in every segment of the alimentary canal, its cellular and subcellular expression in the small intestine were quite distinct from those seen in other segments. In contrast to the stomach and colon, where staining was polarized and restricted to the basolateral surfaces, and in contrast to the epithelial cells of the esophagus and submucosal leukocytes, which showed nonpolarized staining around the entire plasma membrane, the staining in small intestine was mainly intracellular and perinuclear, limited to cells in deep crypts. Prior genetic evidence suggested that a defective HLA-H protein is the molecular basis of HH. Here we show that the HLA-H protein not only varies in its pattern of expression along the cranial/caudal axis of the gastrointestinal tract but that it has a unique subcellular localization in the crypts of the small intestine in proximity to the presumed sites of iron absorption.
Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine
Authors: Bronte Vincenzo , Carroll Miles W. , Goletz Theresa J. , Wang Michael , Overwijk Willem W. , Marincola Francesco , Rosenberg Steven A. , Moss Bernard , Restifo Nicholas P.
Published: July 2002

Recombinant poxviruses encoding tumor-associated antigens (TAA) are attractive as candidate cancer vaccines. Their effectiveness, however, will depend upon expression of the TAA in appropriate antigen-presenting cells. We have used a murine model in which the TAA is β-galactosidase (β-gal) and a panel of recombinant vaccinia viruses (rVV) in which β-gal was expressed under early or late promoters at levels that varied over 500-fold during productive infections in tissue culture cells. Remarkably, only those rVV employing early promoters were capable of prolonging the survival of mice bearing established tumors expressing the model TAA. Late promoters were ineffective regardless of their determined promoter strength. The best results were obtained when β-gal was regulated by a strong early promoter coupled to a strong late promoter. When a variety of cell types were infected with the panel of virusesin vitro, dendritic cells were found to express β-gal only under the control of the early promoters even though late promoters were intrinsically more active in other cell types. Furthermore, in a functional assay, dendritic cells infectedin vitrowith rVV encoding β-gal regulated by an early promoter activated β-gal-specific cytotoxic T lymphocytes, whereas similar rVV with a late promoter-regulated gene did not. These data indicate that promoter strengthper seis not the most critical quality of a recombinant poxvirus-based tumor vaccine and that the use of promoters capable of driving the production of TAA in “professional” antigen presenting cells may be crucial.
Cloning and characterization of human karyopherin β3
Authors: Yaseen Nabeel R. , Blobel Günter
Published: July 2002

Nuclear import of classical nuclear localization sequence-bearing proteins is mediated by karyopherin α/β1 heterodimers. A second nuclear import pathway, mediated by karyopherin β2 (transportin), recently was described for mRNA-binding proteins. Here we report the cloning and characterization of human karyopherin β3, which may be involved in a third pathway for nuclear import. Karyopherin β3 was localized mainly to the cytosol and the nucleus, particularly the nuclear rim. It bound to several of the repeat-containing nucleoporins (Nup358, Nup214, Nup153, Nup98, and p62) in overlay and solution-binding assays and was competed away by karyopherin β1. For Nup98, we localized this binding to the peptide repeat-containing region. Karyopherin β3 contains two putative Ran-binding homology regions and bound to Ran-GTP in a solution-binding assay with much higher affinity than to Ran-GDP. Furthermore, it interacted with two ribosomal proteins in an overlay assay. We suggest that karyopherin β3 is a nuclear transport factor that may mediate the import of some ribosomal proteins into the nucleus.
Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in
Tetrahymena
Authors: Wei Yi , Mizzen Craig A. , Cook Richard G. , Gorovsky Martin A. , Allis C. David
Published: July 2002

H3 phosphorylation has been correlated with mitosis temporally in mammalian cells and spatially in ciliated protozoa. In logarithmically growing
Tetrahymena thermophila
cells, for example, H3 phosphorylation can be detected in germline micronuclei that divide mitotically but not in somatic macronuclei that divide amitotically. Here, we demonstrate that micronuclear H3 phosphorylation occurs at a single site (Ser-10) in the amino-terminal domain of histone H3, the same site phosphorylated during mitosis in mammalian cells. Using an antibody specific for Ser-10 phosphorylated H3, we show that, in
Tetrahymena
, this modification is correlated with mitotic and meiotic divisions of micronuclei in a fashion that closely coincides with chromosome condensation. Our data suggest that H3 phosphorylation at Ser-10 is a highly conserved event among eukaryotes and is likely involved in both mitotic and meiotic chromosome condensation.
The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop
Authors: Ross Ted M. , Cullen Bryan R.
Published: July 2002

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.
Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate
Authors: Goldman Corey K. , Kendall Richard L. , Cabrera Gustavo , Soroceanu Liliana , Heike Yuji , Gillespie G. Yancey , Siegal Gene P. , Mao Xianzhi , Bett Andrew J. , Huckle William R. , Thomas Kenneth A. , Curiel David T.
Published: July 2002

Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the
sflt-1
gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis
in vivo
, since it did not affect tumor cell mitogenesis
in vitro
. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that
sflt-1
gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.
Mast cells mediate acute inflammatory responses to implanted biomaterials
Authors: Tang Liping , Jennings Timothy A. , Eaton John W.
Published: July 2002

Implanted biomaterials trigger acute and chronic inflammatory responses. The mechanisms involved in such acute inflammatory responses can be arbitrarily divided into phagocyte transmigration, chemotaxis, and adhesion to implant surfaces. We earlier observed that two chemokines—macrophage inflammatory protein 1α/monocyte chemoattractant protein 1—and the phagocyte integrin Mac-1 (CD11b/CD18)/surface fibrinogen interaction are, respectively, required for phagocyte chemotaxis and adherence to biomaterial surfaces. However, it is still not clear how the initial transmigration of phagocytes through the endothelial barrier into the area of the implant is triggered. Because implanted biomaterials elicit histaminic responses in the surrounding tissue, and histamine release is known to promote rapid diapedesis of inflammatory cells, we evaluated the possible role of histamine and mast cells in the recruitment of phagocytes to biomaterial implants. Using i.p. and s.c. implantation of polyethylene terephthalate disks in mice we find: (i) Extensive degranulation of mast cells, accompanied by histamine release, occurs adjacent to short-term i.p. implants. (ii) Simultaneous administration of H1 and H2 histamine receptor antagonists (pyrilamine and famotidine, respectively) greatly diminishes recruitment and adhesion of both neutrophils (<20% of control) and monocytes/macrophages (<30% of control) to implants. (iii) Congenitally mast cell-deficient mice also exhibit markedly reduced accumulation of phagocytes on both i.p. and s.c implants. (iv) Finally, mast cell reconstitution of mast cell-deficient mice restores “normal” inflammatory responses to biomaterial implants. We conclude that mast cells and their granular products, especially histamine, are important in recruitment of inflammatory cells to biomaterial implants. Improved knowledge of such responses may permit purposeful modulation of both acute and chronic inflammation affecting implanted biomaterials.
Spatiotemporal imaging of human brain activity using functional MRI constrained magnetoencephalography data: Monte Carlo simulations
Authors: Liu Arthur K. , Belliveau John W. , Dale Anders M.
Published: July 2002

The goal of our research is to develop an experimental and analytical framework for spatiotemporal imaging of human brain function. Preliminary studies suggest that noninvasive spatiotemporal maps of cerebral activity can be produced by combining the high spatial resolution (millimeters) of functional MRI (fMRI) with the high temporal resolution (milliseconds) of electroencephalography (EEG) and magnetoencephalography (MEG). Although MEG and EEG are sensitive to millisecond changes in mental activity, the ability to resolve source localization and timing is limited by the ill-posed “inverse” problem. We conducted Monte Carlo simulations to evaluate the use of MRI constraints in a linear estimation inverse procedure, where fMRI weighting, cortical location and orientation, and sensor noise statistics were realistically incorporated. An error metric was computed to quantify the effects of fMRI invisible (“missing”) sources, “extra” fMRI sources, and cortical orientation errors. Our simulation results demonstrate that prior anatomical and functional information from MRI can be used to regularize the EEG/MEG inverse problem, giving an improved solution with high spatial and temporal resolution. An fMRI weighting of approximately 90% was determined to provide the best compromise between separation of activity from correctly localized sources and minimization of error caused by missing sources. The accuracy of the estimate was relatively independent of the number and extent of the sources, allowing for incorporation of physiologically realistic multiple distributed sources. This linear estimation method provides an operator-independent approach for combining information from fMRI, MEG, and EEG and represents a significant advance over traditional dipole modeling.
Synaptic signaling in an active central network only moderately changes passive membrane properties
Authors: Raastad Morten , Enríquez-Denton Manuel , Kiehn Ole
Published: July 2002

The membrane resistance of mammalian central neurons may be dramatically reduced by synaptic events during network activity, thereby changing their integration properties. We have used the isolated neonatal rat spinal cord to provide measurements of the effect of synaptic signaling on passive membrane properties during network activity. Synaptic signaling could take place during fictive locomotor activity with only modest (on average 35%) reduction of the input resistance (
R
in
) and of the cell’s charging time constant (τ
in
). Individual synaptic signals, however, often introduced a peak conductance that was greater than the input conductance (
G
in
= 1
/R
in
) without synaptic activity. The combination of moderate average synaptic conductance and large conductance of individual synaptic signals suggests that individual presynaptic neurons have large but short-lasting influence on the integration properties of postsynaptic neurons.
Increase of intracellular Ca
2+
and relocation of E-cadherin during experimental decompaction of mouse embryos
Authors: Pey Roxana , Vial Clarisa , Schatten Gerald , Hafner Mathias
Published: July 2002

To determine the role of intracellular Ca
2+
in compaction, the first morphogenetic event in embryogenesis, we analyzed preimplantation mouse embryos under several decompacting conditions, including depletion of extracellular Ca
2+
, blocking of Ca
2+
channels, and inhibition of microfilaments, calmodulin, and intracellular Ca
2+
release. Those treatments induced decompaction of mouse morulae and simultaneously induced changes in cytosolic free Ca
2+
concentration and deregionalization of E-cadherin and fodrin. When morulae were allowed to recompact, the location of both proteins recovered. In contrast, actin did not change its cortical location with compaction nor with decompaction-recompaction. Calmodulin localized in areas opposite to cell–cell contacts in eight-cell stage embryos before and after compaction. Inhibition of calmodulin with trifluoperazine induced its delocalization while morulae decompacted. A nonspecific rise of intracellular free Ca
2+
provoked by ionomycin did not affect the compacted shape. Moreover, the same decompacting treatments when applied to uncompacted embryos did not produce any change in intracellular Ca
2+
. Our results demonstrate that in preimplantation mouse embryos experimentally induced stage-specific changes of cell shape are accompanied by changes of intracellular free Ca
2+
and redistribution of the cytoskeleton-related proteins E-cadherin, fodrin, and calmodulin. We conclude that intracellular Ca
2+
specifically is involved in compaction and probably regulates the function and localization of cytoskeleton elements.
Taxane-mediated gene induction is independent of microtubule stabilization: Induction of transcription regulators and enzymes that modulate inflammation and apoptosis
Authors: Moos Philip J. , Fitzpatrick F. A.
Published: July 2002

Pharmacological traits of the antineoplastic agent taxol may originate in part from its effects on gene expression and not simply from its effects on microtubule assembly. This prompts three questions. First, how extensive is gene induction by taxol? Second, is gene induction confined to taxol itself, or does it occur with other taxane analogs? Third, do the functions of any induced genes correspond with known attributes of taxol or taxane analogs? We report that taxol induces numerous early-response genes, not just cytokine genes. Previously unidentified taxol-induced genes include genes coding transcription factors with tumor suppressor effects (krox-24) and enzymes that govern proliferation, apoptosis, and inflammation (2′5′-oligoadenylate synthase,cyclooxygenase-2, and an IκB kinase termedchuk). Taxotere, a potent analog of taxol, did not induce any of these genes, implying that taxol modulates gene expression by a mechanism that is distinct from microtubule stabilization and cell cycle arrest. Other taxane analogs induce some of the same genes as taxol, indicating that this process is not unique to taxol. Functional changes coincided with changes in gene expression. For instance, induction of tumor necrosis factor α (TNFα) accentuated apoptosis in cells treated with taxol compared with corresponding cells treated with taxotere. The functions of several induced genes (e.g.,krox-24andcyclooxygenase-2) are self-consistent with beneficial and adverse effects encountered during taxol administration. These results may be relevant to the safe and effective use of taxol or its analogs in oncology and other areas of medicine.
Heterothermal acclimation: An experimental paradigm for studying the control of thermal acclimation in crabs
Authors: Cuculescu Mirela , Pearson Timothy , Hyde David , Bowler Ken
Published: July 2002

A method for the study of the control of the attainment of thermal acclimation has been applied to the crabs,
Cancer pagurus
and
Carcinus maenas
. Crabs were heterothermally acclimated by using an anterior–posterior partition between two compartments, one at 8°C and the other at 22°C. One compartment held a three-quarter section of the crab including the central nervous system (CNS), eye stalks, and ipsilateral legs; the other held a quarter section including the contralateral legs. Criteria used to assess the acclimation responses were comparisons of muscle plasma membrane fatty acid composition and “fluidity.” In both species, the major fatty acids of phosphatidylcholine were 16:0, 18:1, 20:5, and 22:6, whereas phosphatidylethanolamine contained significantly less 16:0 but more 18:0; these fatty acids comprised 80% of the total. Differences in fatty acid composition were demonstrated between fractions obtained from the ipsilateral and contralateral legs from the same heterothermally acclimated individual. In all acclimation states (except 22CNS, phosphatidylcholine fraction), membrane lipid saturation was significantly increased with acclimation at 22° as compared with 8°C. Membrane fluidity was determined by using 1,3-diphenyl-1,3,5 hexatriene (DPH) fluorescence polarization. In both species, membranes from legs held at 8° were more fluid than from legs held at 22°C irrespective of the acclimation temperature of the CNS. Heterothermal acclimation demonstrated that leg muscle membrane composition and fluidity respond primarily to local temperature and were not predominately under central direction. The responses between 8°C- and 22°C-acclimated legs were more pronounced when the CNS was cold-acclimated, so a central influence cannot be excluded.
Disruption of
mRad50
causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation
Authors: Luo Guangbin , Yao Michelle S. , Bender Carla F. , Mills Michael , Bladl Anthony R. , Bradley Allan , Petrini John H. J.
Published: July 2002

The Mre11/Rad50 protein complex functions in diverse aspects of the cellular response to double-strand breaks (DSBs), including the detection of DNA damage, the activation of cell cycle checkpoints, and DSB repair. Whereas genetic analyses in
Saccharomyces cerevisiae
have provided insight regarding DSB repair functions of this highly conserved complex, the implication of the human complex in Nijmegen breakage syndrome reveals its role in cell cycle checkpoint functions. We established
mRad50
mutant mice to examine the role of the mammalian Mre11/Rad50 protein complex in the DNA damage response. Early embryonic cells deficient in
mRad50
are hypersensitive to ionizing radiation, consistent with a role for this complex in the repair of ionizing radiation-induced DSBs. However, the null
mrad50
mutation is lethal in cultured embryonic stem cells and in early developing embryos, indicating that the mammalian Mre11/Rad50 protein complex mediates functions in normally growing cells that are essential for viability.
Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the
ilvYC
operon of
Escherichia coli
Authors: Rhee Kyu Y. , Opel Michael , Ito Elaine , Hung She-pin , Arfin Stuart M. , Hatfield G. Wesley
Published: July 2002

The twin-domain model [Liu, L. F. & Wang, J. C. (1987)
Proc. Natl. Acad. Sci. USA
84, 7024–7027] suggests that closely spaced, divergent, superhelically sensitive promoters can affect the transcriptional activity of one another by transcriptionally induced negative DNA supercoiling generated in the divergent promoter region. This gene arrangement is observed for many LysR-type-regulated operons in bacteria. We have examined the effects of divergent transcription in the prototypic LysR-type system, the
ilvYC
operon of
Escherichia coli
. Double-reporter constructs with the
lacZ
gene under transcriptional control of the
ilvC
promoter and the
galK
gene under control of the divergent
ilvY
promoter were used to demonstrate that a down-promoter mutation in the
ilvY
promoter severely decreases
in vivo
transcription from the
ilvC
promoter. However, a down-promoter mutation in the
ilvC
promoter only slightly affects transcription from the
ilvY
promoter.
In vitro
transcription assays with DNA topoisomers showed that transcription from the
ilvC
promoter increases over the entire range of physiological superhelical densities, whereas transcription initiation from the
ilvY
promoter exhibits a broad optimum at a midphysiological superhelical density. Evidence that this promoter coupling is DNA supercoiling-dependent is provided by the observation that a novobiocin-induced decrease in global negative superhelicity results in an increase in
ilvY
promoter activity and a decrease in
ilvC
promoter activity predicted by the
in vitro
data. We suggest that this transcriptional coupling is important for coordinating basal level expression of the
ilvYC
operon with the nutritional and environmental conditions of cell growth.
Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus
Authors: Wilson Steven P. , Yeomans David C. , Bender Mary Ann , Lu Ying , Goins William F. , Glorioso Joseph C.
Published: July 2002

To test the utility of gene therapeutic approaches for the treatment of pain, a recombinant herpes simplex virus, type 1, has been engineered to contain the cDNA for an opioid peptide precursor, human preproenkephalin, under control of the human cytomegalovirus promoter. This virus and a similar recombinant containing the
Escherichia coli lacZ
gene were applied to the abraded skin of the dorsal hindpaw of mice. After infection, the presence of β-galactosidase in neuronal cell bodies of the relevant spinal ganglia (
lacZ
-containing virus) and of human proenkephalin (preproenkephalin-encoding virus) in the central terminals of these neurons indicated appropriate gene delivery and expression. Baseline foot withdrawal responses to noxious radiant heat mediated by Aδ and C fibers were similar in animals infected with proenkephalin-encoding and β-galactosidase-encoding viruses. Sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (Aδ fibers) observed in control animals was reduced or eliminated in animals infected with the proenkephalin-encoding virus for at least 7 weeks postinfection. Hence, preproenkephalin cDNA delivery selectively blocked hyperalgesia without disrupting baseline sensory neurotransmission. This blockade of sensitization was reversed by administration of the opioid antagonist naloxone, apparently acting in the spinal cord. The results demonstrate that the function of sensory neurons can be selectively altered by viral delivery of a transgene. Because hyperalgesic mechanisms may be important in establishing and maintaining neuropathic and other chronic pain states, this approach may be useful for treatment of chronic pain and hyperalgesia in humans.
Structural interactions of fibroblast growth factor receptor with its ligands
Authors: Stauber Deborah J. , DiGabriele Anna D. , Hendrickson Wayne A.
Published: July 2002

Fibroblast growth factors (FGFs) effect cellular responses by binding to FGF receptors (FGFRs). FGF bound to extracellular domains on the FGFR in the presence of heparin activates the cytoplasmic receptor tyrosine kinase through autophosphorylation. We have crystallized a complex between human FGF1 and a two-domain extracellular fragment of human FGFR2. The crystal structure, determined by multiwavelength anomalous diffraction analysis of the selenomethionyl protein, is a dimeric assemblage of 1:1 ligand:receptor complexes. FGF is bound at the junction between the two domains of one FGFR, and two such units are associated through receptor:receptor and secondary ligand:receptor interfaces. Sulfate ion positions appear to mark the course of heparin binding between FGF molecules through a basic region on receptor D2 domains. This dimeric assemblage provides a structural mechanism for FGF signal transduction.
Song selectivity and sensorimotor signals in vocal learning and production
Authors: Solis Michele M. , Brainard Michael S. , Hessler Neal A. , Doupe Allison J.
Published: July 2002

Bird song, like human speech, is a learned vocal behavior that
requires auditory feedback. Both as juveniles, while they learn to
sing, and as adults, songbirds use auditory feedback to compare their
own vocalizations with an internal model of a target song. Here we
describe experiments that explore a role for the songbird anterior
forebrain pathway (AFP), a basal ganglia-forebrain circuit, in
evaluating song feedback and modifying vocal output. First, neural
recordings in anesthetized, juvenile birds show that single AFP neurons
are specialized to process the song stimuli that are compared during
sensorimotor learning. AFP neurons are tuned to both the bird's own
song and the tutor song, even when these stimuli are manipulated to be
very different from each other. Second, behavioral experiments in adult
birds demonstrate that lesions to the AFP block the deterioration of
song that normally follows deafening. This observation suggests that
deafening results in an instructive signal, indicating a mismatch
between feedback and the internal song model, and that the AFP is
involved in generating or transmitting this instructive signal.
Finally, neural recordings from behaving birds reveal robust
singing-related activity in the AFP. This activity is likely to
originate from premotor areas and could be modulated by auditory
feedback of the bird's own voice. One possibility is that this
activity represents an efference copy, predicting the sensory
consequences of motor commands. Overall, these studies illustrate that
sensory and motor processes are highly interrelated in this circuit
devoted to vocal learning, as is true for brain areas involved in
speech.
Polymerization of nontemplate bases before transcription initiation at the 3′ ends of templates by an RNA-dependent RNA polymerase: An activity involved in 3′ end repair of viral RNAs
Authors: Guan Hancheng , Simon Anne E.
Published: July 2002

The 3′ ends of RNAs associated with turnip crinkle virus (TCV),
including subviral satellite (sat)C, terminate with the motif
CCUGCCC-3′. Transcripts of satC with a deletion of the motif are
repaired to wild type (wt)
in vivo
by RNA-dependent RNA
polymerase (RdRp)-mediated extension of abortively synthesized
oligoribonucleotide primers complementary to the 3′ end of the TCV
genomic RNA. Repair of shorter deletions, however, are repaired by
other mechanisms. SatC transcripts with the 3′ terminal CCC replaced by
eight nonviral bases were repaired in plants by homologous
recombination between the similar 3′ ends of satC and TCV. Transcripts
with deletions of four or five 3′ terminal bases, in the presence or
absence of nonviral bases, generated progeny with a mixture of wt and
non-wt 3′ ends
in vivo
.
In vitro
,
RdRp-containing extracts were able to polymerize nucleotides in a
template-independent fashion before using these primers to initiate
transcription at or near the 3′ end of truncated satC templates. The
nontemplate additions at the 5′ ends of the nascent complementary
strands were not random, with a preference for consecutive identical
nucleotides. The RdRp was also able to initiate transcription opposite
cytidylate, uridylate, guanylate, and possibly adenylate residues
without exhibiting an obvious preference, flexibility previously
unreported for viral RdRp. The unexpected existence of three different
repair mechanisms for TCV suggests that 3′ end reconstruction is
critical to virus survival.
Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in theplt/pltmouse
Authors: Luther Sanjiv A. , Tang H. Lucy , Hyman Paul L. , Farr Andrew G. , Cyster Jason G.
Published: July 2002

The spontaneous mutant mouse strain,plt/plt, lacks the secondary lymphoid organ chemokine (SLC)-ser gene and has disrupted trafficking of T cells and dendritic cells (DCs) to lymphoid tissues. We demonstrate here that the gene for the related chemokine, Epstein–Barr virus-induced molecule-1 ligand chemokine (ELC), is also deleted in this immunodeficient mouse strain. Using a combination of approaches, including bone marrow reconstitution and doublein situhybridization, we show in wild-type mice that ELC is expressed by T zone stromal cells that also make SLC. Smaller amounts of ELC are made by DCs, predominantly of the CD8+phenotype. We propose that ELC- and SLC-expressing T zone stromal cells play a central role in bringing naive T cells and DCs together for the initiation of immune responses.
Consciousness and Commentaries
Authors: Weiskrantz L.
Published: July 2002

There are several neuropsychological syndromes in which good residual function is retained in the absence of acknowledged awareness, among them blindsight, amnesia, and unilateral neglect. All of them point to the need in studying conscious awareness not only for an “on‐line” demonstration of the relevant capacity, but for an independent commentary or classificatory response by the subject, whether human or animal. The parametric limits of blindsight (visual discrimination without awareness) can be measured using a “commentary key” psychophysical paradigm, and the results may possibly allow an approach to identifying neural structures involved in visual awareness.
“Ethnic Conflict” Undressed: Patterns of Contrast, Interest of Elites, and Clientelism of Foreign Powers in Comparative Perspective—Bosnia, India, Pakistan
Authors: Wieland Carsten
Published: July 2002

Ethnic conflict is not—because there are no ethnic groups in conflict. This is the main conclusion of a comparison of so-called “ethnic conflicts” in the Balkans and in colonial India. A comparison of Muslim nation building in these two regions provides several valuable insights that go far beyond the specific cases. Thus far, there have been many hints in the literature on similarities between Bosnia and Pakistan or the Balkans and the Indian subcontinent as a whole. But there have been no systematic comparisons, though many parallels emerge when we look more closely.
The prevalence and management of co‐morbid substance misuse and mental illness: results of a screening survey in substance misuse and mental health treatment populations
Authors: WEAVER TIM , HICKMAN MATTHEW , RUTTER DEBORAH , WARD JENNI , STIMSON GERRY , RENTON ADRIAN
Published: July 2002

A cross‐sectional survey of clinical staff was carried out among statutory (public sector) substance misuse services and community mental health teams (CMHTs) in an inner London borough. We identified 368 current patients of drug and alcohol misuse services and 930 current patients of CMHTs. We measured the prevalence of co‐morbid substance misuse and mental illness among these patients. We found strong evidence of co‐morbid mental health problems in 128 substance misuse patients (34.8%) and evidence of possible co‐morbidity in a further 18.5% of cases. Among mental health service patients, 227 (24%) were reported to have current substance misuse problems. Shared care of patients was uncommon and only a minority of co‐morbid patients received treatment for both problems or the specialist assessment which key workers felt they needed. The findings suggest that co‐morbidity is highly prevalent among mental health and substance misuse patients. Further research is needed to corroborate these findings, but the development and provision of evidence‐based treatment and service interventions is likely to become an increasing priority.
Prevalence of drug use prior to detention among residents of youth detention centres in Queensland
Authors: LENNINGS CHRISTOPHER , PRITCHARD MONICA
Published: July 2002

Research into juvenile delinquency has established a strong connection between delinquent behaviour and drug usage. Anecdotal evidence suggests that a number of young people coming into detention in Queensland are suffering physical, behavioural and emotional consequences of their drug use prior to detention. Between December 1995 and January 1996, a total of 118 young people in detention were surveyed regarding their drug use behaviour prior to detention. This represented 90% of the average total population in detention. The average age of the sample was 15.6 years; 90% were males. Over 90% of the population had used (or at least tried) pain relievers, other medicines, cannabis, alcohol and tobacco. The use of the so called ‘hard drugs’ was high, with 47% having tried amphetamines, 35% admitting to having tried heroin, and 64% having used hallucinogens at least once. Data regarding harm‐minimization practices revealed that 42% had injected drugs and of these half had shared a needle at least once, while around 90% are sexually active and of these only about 20% always use a condom.
Book Reviews
Authors: GOSSOP MICHAEL
Published: July 2002

Books Reviewed in this Article:

Source Book of Substance Abuse and Addiction LAWRENCE FRIEDMAN, NICHOLAS FLEMING, DAVID ROBERTS & STEVEN HYMAN (Eds)

Methadone in the Treatment of Narcotic Addiction ANDREW BYRNE

Policing Places with Drug Problems Vol. 2, Drugs, health, and social policy LORRAINE GREEN

Alcohol Problems among Adolescents BOYD, HOWARD & ZUCKER (Eds)

Dying For a Drink: a no‐nonsense guide for heavy drinkers TIM CANTOPHER

Addictive Behaviour: molecules to mankind –perspectives on the nature of addiction A. BONNER & J. WATERHOUSE (Eds)
Regulation of Neural Cell Adhesion Molecule Polysialylation: Evidence for Nontranscriptional Control and Sensitivity to an Intracellular Pool of Calcium
Authors: Brusés Juan L. , Rutishauser Urs
Published: July 2002

The up- and downregulation of polysialic acid–neural cell adhesion molecule (PSA–NCAM) expression on motorneurons during development is associated respectively with target innervation and synaptogenesis, and is regulated at the level of PSA enzymatic biosynthesis involving specific polysialyltransferase activity. The purpose of this study has been to describe the cellular mechanisms by which that regulation might occur. It has been found that developmental regulation of PSA synthesis by ciliary ganglion motorneurons is not reflected in the levels of polysialyltransferase-1 (PST) or sialyltransferase-X (STX) mRNA. On the other hand, PSA synthesis in both the ciliary ganglion and the developing tectum appears to be coupled to the concentration of calcium in intracellular compartments. This study documents a calcium dependence of polysialyltransferase activity in a cell-free assay over the range of 0.1–1 mM, and a rapid sensitivity of new PSA synthesis, as measured in a pulse–chase analysis of tissue explants, to calcium ionophore perturbation of intracellular calcium levels. Moreover, the relevant calcium pool appears to be within a specific intracellular compartment that is sensitive to thapsigargin and does not directly reflect the level of cytosolic calcium. Perturbation of other major second messenger systems, such as cAMP and protein kinase–dependent pathways, did not affect polysialylation in the pulse chase analysis. These results suggest that the shuttling of calcium to different pools within the cell can result in the rapid regulation of PSA synthesis in developing tissues.
The “8-kD” Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans
Authors: Beckwith Susan M. , Roghi Christian H. , Liu Bo , Ronald Morris N.
Published: July 2002

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the “8-kD” cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.
Autocrine Tumor Necrosis Factor (TNF) and Lymphotoxin (LT) α Differentially Modulate Cellular Sensitivity to TNF/LT-α Cytotoxicity in L929 Cells
Authors: Decoster Els , Cornelis Sigrid , Vanhaesebroeck Bart , Fiers Walter
Published: July 2002

Tumor necrosis factor (TNF) and lymphotoxin (LT) α are structurally and functionally related cytokines. We expressed the TNF and LT-α genes in murine fibrosarcoma L929r2 cells, which can be sensitized to TNF/LT-α–dependent necrosis by inhibitors of transcription or translation. Autocrine production of murine TNF in L929r2 cells completely downmodulated the expression of the 55- and 75-kD TNF receptors, resulting in resistance to TNF/LT-α cytotoxicity. Partial downmodulation of the 55-kD receptor was observed in human TNF-producing L929r2 cells. In contrast, an unaltered TNF receptor expression was found on LT-α L929r2 transfectants. Hence, although similar cytotoxic effects are induced by extracellularly administered TNF and LT-α, endogenous expression of these cytokines fundamentally differs in the way they modulate TNF receptor expression. Unlike LT-α, secreted by the classical pathway, TNF is first formed as a membrane-bound protein, which is responsible for receptor downmodulation. To explore whether the different pathways for secretion of TNF and LT-α explain this difference, we examined the effect of membrane-bound LT-α expression. This was obtained by exchange of the classical signal sequence of LT-α for the membrane anchor of chicken hepatic lectin. Membrane retention of LT-α resulted indeed in receptor downmodulation and TNF/LT-α resistance. We conclude that membrane retention of newly synthesized TNF or LT-α is absolutely required for receptor downmodulation and TNF/LT-α resistance.
Phosphorylation-dependent Binding of Hepatitis B Virus Core Particles to the Nuclear Pore Complex
Authors: Kann Michael , Sodeik Beate , Vlachou Angelika , Gerlich Wolfram H. , Helenius Ari
Published: July 2002

Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) α and β. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.
Regulation of Cell Contraction and Membrane Ruffling by Distinct Signals in Migratory Cells
Authors: Cheresh David A. , Leng Jie , Klemke Richard L.
Published: July 2002

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.
Peroxisomal Membrane Fusion Requires Two Aaa Family Atpases, Pex1p and Pex6p
Authors: Titorenko Vladimir I. , Rachubinski Richard A.
Published: July 2002

Two AAA family ATPases, NSF and p97, have been implicated in membrane fusion during assembly and inheritance of organelles of the secretory pathway. We have now investigated the roles of AAA ATPases in membrane fusion during assembly of the peroxisome, an organelle outside the classical secretory system. Here, we show that peroxisomal membrane fusion in the yeast Yarrowia lipolytica requires two AAA ATPases, Pex1p and Pex6p. Release of membrane- associated Pex1p and Pex6p drives the asymmetric priming of two fusion partners. The next step, peroxisome docking, requires release of Pex1p from one partner. Subsequent fusion of the peroxisomal membranes is independent of both Pex1p and Pex6p.