bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA-dependent phosphorylation of a nuclear RNA binding protein
Authors: Fung Peter A. , Labrecque Roxanne , Pederson Thoru
Published: July 2002

The human C1 heterogeneous nuclear ribonucleoprotein particle protein (hnRNP protein) undergoes a cycle of phosphorylation–dephosphorylation in HeLa cell nuclear extracts that modulates the binding of this protein to pre-mRNA. We now report that hyperphosphorylation of the C1 hnRNP protein is mediated by a kinase activity in nuclear extracts that is RNA-dependent. Although the basal phosphorylation of the C1 hnRNP protein in nuclear extracts reflects a casein kinase II-type activity, its RNA-dependent hyperphosphorylation appears to be mediated by a different kinase. This is indicated by the unresponsiveness of the RNA-stimulated hyperphosphorylation to casein kinase II inhibitors, and the distinct glycerol gradient sedimentation profiles of the basal versus RNA-stimulated C1 hnRNP protein phosphorylation activities from nuclear extracts. RNA-dependent phosphorylation was observed both for a histidine-tagged recombinant human C1 hnRNP protein added to nuclear extracts and also for the endogenous C1 hnRNP protein. Additional results rule out protein kinase A, protein kinase C, calmodulin-dependent protein kinase II, and double-stranded RNA-activated protein kinase as the enzymes responsible for the RNA-dependent hyperphosphorylation of the C1 hnRNP protein. These results reveal the existence in nuclear extracts of an RNA-dependent protein kinase activity that hyperphosphorylates a known pre-mRNA binding protein, and define an additional element to be integrated into the current picture of how nuclear proteins are regulated by phosphorylation.
Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates
Authors: Schmalzing Günther , Ruhl Karina , Gloor Sergio M.
Published: July 2002

The functional unit of the Na,K-ATPase consists of a catalytic α subunit noncovalently linked with a glycoprotein subunit, β. Using ouabain binding assays and immunoprecipitation of rodent α/β complexes, we show here that all six possible isozymes between three α and two β isoforms can be formed inXenopusoocytes. Two isoform-specific differences in α/β interactions are observed: (i) α1/β1 and α2/β2 complexes, in contrast to α1/β2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) β2 subunits must carryN-glycans to combine with α1 but not with α2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated β1 subunit comprising the ectodomain results in assembly with α1 and α2, but not with α3; the β2 ectodomain combines with α2 only. A chimera consisting of 81% and 19% of the α1 N terminus and α2 C terminus, respectively, behaves like α2 and coprecipitates with the β2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the β2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase α and β subunits.
Restrictive clonal allocation in the chimeric mouse brain
Authors: Kuan Chia-Yi , Elliott Eileen A. , Flavell Richard A. , Rakic Pasko
Published: July 2002

Whether, and to what extent, lineage restriction contributes to the organization of the mammalian brain remains unclear. Here we address this issue by examining the distribution of clonally related cells in chimeric mice generated by injecting genetically tagged embryonic stem (ES) cells into blastocyst embryos. Our examination of postnatal chimeric brains revealed that the vast majority of labeled ES cell descendents were confined within a different subset of brain regions in each animal. Moreover, the deployment of labeled cells in different brain regions was distinctive. The pattern of ordered and binomial colonization suggested that early diversified founder cells may constrain the fates of their descendants through a restriction of dispersion. In addition, the symmetrical distribution of ES cell descendants suggests that bilaterally corresponding structures may arise from a common set of progenitor cells. Finally, clones of cells formed a continuous band within the deep strata of the neocortex. This later finding in conjunction with the radial distribution of clones in remaining layers observed in previous studies indicates that the cerebral neocortex may derive from two groups of founder cells, which is consistent with the hypothesis of dual phylogenetic origins of the mammalian cerebral cortex.
RhoGDIγ: A GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas
Authors: Adra Chaker N. , Manor Danny , Ko Jon L. , Zhu Shaochun , Horiuchi Tadashi , Van Aelst Linda , Cerione Richard A. , Lim Bing
Published: July 2002

GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activation of GTPases and may also be critical for the cellular compartmentalization of GTPases. RhoGDI and GDI/D4 are two currently known GDIs for the Rho-subfamily of GTPases. Using their cDNAs to screen a human brain cDNA library under low stringency, we have cloned a homologous cDNA preferentially expressed at high levels in brain and pancreas. The predicted protein, named RhoGDIγ, is ≈50% identical to GDI/D4 and RhoGDI. It binds to CDC42 and RhoA with less affinity compared with RhoGDI and does not bind with Rac1, Rac2, or Ras. RhoGDIγ functions as a GDI for CDC42 but with ≈20 times less efficiency than RhoGDI. Immunohistochemical studies showed a diffuse punctate distribution of the protein in the cytoplasm with concentration around the nucleus in cytoplasmic vesicles. Overexpression of the protein in baby hamster kidney cells caused the cells to round up with loss of stress fibers. A distinct hydrophobic amino terminus in RhoGDIγ, not seen in the other two RhoGDIs, could provide a mechanism for localization of the GDI to specific membranous compartment thus determining function distinct from RhoGDI or GDI/D4. Our results provide evidence that there is a family of GDIs for the Rho-related GTPases and that they differ in binding affinity, target specificity, and tissue expression. We propose that RhoGDI be renamed RhoGDIα and GDID4 be renamed RhoGDIβ. The new GDI should widen the scope of investigation of this important class of regulatory protein.
A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: A novel defect in plasmalogen biosynthesis
Authors: Nagan Narasimhan , Hajra Amiya K. , Das Arun K. , Moser Hugo W. , Moser Ann , Lazarow Paul , Purdue P. Edward , Zoeller Raphael A.
Published: July 2002

Using fluorescence-activated cytotoxicity selection, followed by colony autoradiographic screening of the surviving population, we have isolated a unique plasmalogen-deficient Chinese hamster ovary (CHO) cell line. The mutant, NZel-1, showed a dramatic (90%) reduction in the rate of biosynthesis and levels of plasmalogens, as determined using short- and long-term labeling with
32
P
i
. Enzymatic assays and lipid supplementation studies showed that NZel-1 was defective in a single step in the biosynthetic pathway for plasmalogens. This step, catalyzed by the peroxisomal enzyme, alkyl-dihydroxyacetone phosphate (DHAP) synthase, is responsible for the introduction of the ether bond found in plasmalogens. The activity of alkyl-DHAP synthase was reduced in whole-cell homogenates from NZel-1 to 18% of wild-type values. Unlike previously described plasmalogen-deficient mutants, NZel-1 contained peroxisomes, as confirmed by immunofluorescence microscopy and catalase release by digitonin. Peroxisomal functions, including the breakdown of very long-chain (>20 carbons) fatty acids, phytanic acid oxidation, and the acylation of DHAP, were normal. Cell fusion studies revealed that the mutation is recessive and belongs to a new complementation group. To our knowledge this is the first report describing the isolation and characterization of a mutant CHO cell line defective in plasmalogen biosynthesis which contains intact, functional peroxisomes. These cells will allow us to examine the role of ether lipids in cellular functions without complications associated with peroxisome deficiency.
The tobacco wounding-activated mitogen-activated protein kinase is encoded by
SIPK
Authors: Zhang Shuqun , Klessig Daniel F.
Published: July 2002

It has been demonstrated that both salicylic acid and fungal elicitors activate a 48-kDa mitogen-activated protein kinase termed salicylic acid-induced protein kinase (SIPK) in tobacco suspension cells. Here, we show that infiltration of these agents into tobacco leaves also activates SIPK. Of particular interest, infiltration of water alone activated a kinase of the same size, possibly because of wounding and/or osmotic stresses. The kinetics of kinase activation, however, differ for these different treatments. Various mechanical stresses, including cutting and wounding by abrasion, also activated a 48-kDa kinase. By using an immune-complex kinase assay with antibodies specific for SIPK or wounding-induced protein kinase, we demonstrate that this wounding-activated 48-kDa kinase is SIPK, rather than wounding-induced protein kinase, as reported [Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. & Ohashi, Y. (1995)
Science
270, 1988–1992]. Activation of SIPK after wounding was associated with tyrosine phosphorylation but not with increases in
SIPK
mRNA or protein levels. Thus, the same mitogen-activated protein kinase, SIPK, appears to facilitate signaling for two distinct pathways that lead to disease resistance responses and wounding responses.
Representation of spatial frequency and orientation in the visual cortex
Authors: Everson R. M. , Prashanth A. K. , Gabbay M. , Knight B. W. , Sirovich L. , Kaplan E.
Published: July 2002

Knowledge of the response of the primary visual cortex to the various spatial frequencies and orientations in the visual scene should help us understand the principles by which the brain recognizes patterns. Current information about the cortical layout of spatial frequency response is still incomplete because of difficulties in recording and interpreting adequate data. Here, we report results from a study of the cat primary visual cortex in which we employed a new image-analysis method that allows improved separation of signal from noise and that we used to examine the neurooptical response of the primary visual cortex to drifting sine gratings over a range of orientations and spatial frequencies. We found that (
i
) the optical responses to all orientations and spatial frequencies were well approximated by weighted sums of only two pairs of basis pictures, one pair for orientation and a different pair for spatial frequency; (
ii
) the weightings of the two pictures in each pair were approximately in quadrature (1/4 cycle apart); and (
iii
) our spatial frequency data revealed a cortical map that continuously assigns different optimal spatial frequency responses to different cortical locations over the entire spatial frequency range.
Neuronal migration disorders: Heterotopic neocortical neurons in CA1 provide a bridge between the hippocampus and the neocortex
Authors: Chevassus-Au-Louis N. , Congar P. , Represa A. , Ben-Ari Y. , Gaïarsa J.-L.
Published: July 2002

Neuronal migration disorders have been involved in various pathologies, including epilepsy, but the properties of the neural networks underlying disorders have not been determined. In the present study, patch clamp recordings were made from intrahippocampal heterotopic as well as from neocortical and hippocampal neurons from brain slices of rats with prenatally methylazoxymethanol-induced cortical malformation. We report that heterotopic neurons have morphometrical parameters and cellular properties of neocortical supragranular neurons and are integrated in both neocortical and hippocampal networks. Thus, stimulation of the white matter induces both antidromic and orthodromic response in heterotopic and neocortical neurons. Stimulation of hippocampal afferents evokes a monosynaptic response in the majority of heterotopic neurons and a polysynaptic all-or-none epileptiform burst in the presence of bicuculline to block γ-aminobutyric acid type A inhibition. Furthermore, hippocampal paroxysmal activity generated by bath application of bicuculline can spread directly to the neocortex via the heterotopia in methylazoxymethanol-treated but not in naive rats. We conclude that heterotopias form a functional bridge between the limbic system and the neocortex, providing a substrate for pathological conditions.
c-Myc target gene specificity is determined by a post-DNAbinding mechanism
Authors: Boyd Kathryn E. , Wells Julie , Gutman Julie , Bartley Stephanie M. , Farnham Peggy J.
Published: July 2002

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that
cad
is a Myc target gene. In addition, we show that Myc binding can occur at many sites
in vivo
but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors
in vivo
to single copy mammalian genes.
Basolateral amygdala is not critical for cognitive memory of contextual fear conditioning
Authors: Vazdarjanova Almira , McGaugh James L.
Published: July 2002

Evidence that lesions of the basolateral amygdala complex (BLC) impair memory for fear conditioning in rats, measured by lack of “freezing” behavior in the presence of cues previously paired with footshocks, has suggested that the BLC may be a critical locus for the memory of fear conditioning. However, evidence that BLC lesions may impair unlearned as well as conditioned freezing makes it difficult to interpret the findings of studies assessing conditioned fear with freezing. The present study investigated whether such lesions prevent the expression of several measures of memory for contextual fear conditioning in addition to freezing. On day 1, rats with sham lesions or BLC lesions explored a Y maze. The BLC-lesioned rats (BLC rats) displayed a greater exploratory activity. On day 2, each of the rats was placed in the “shock” arm of the maze, and all of the sham and half of the BLC rats received footshocks. A 24-hr retention test assessed the freezing, time spent per arm, entries per arm, and initial entry into the shock arm. As previously reported, shocked BLC rats displayed little freezing. However, the other measures indicated that the shocked BLC rats remembered the fear conditioning. They entered less readily and less often and spent less time in the shock arm than did the control nonshocked BLC rats. Compared with the sham rats, the shocked BLC rats entered more quickly and more often and spent more time in the shock arm. These findings indicate that an intact BLC is not essential for the formation and expression of long-term cognitive/explicit memory of contextual fear conditioning.
Early physiological abnormalities after simian immunodeficiency virus infection
Authors: Horn Thomas F. W. , Huitron-Resendiz Salvador , Weed Michael R. , Henriksen Steven J. , Fox Howard S.
Published: July 2002

Central nervous system (CNS) damage and dysfunction are devastating consequences of HIV infection. Although the CNS is one of the initial targets for HIV infection, little is known about early viral-induced abnormalities that can affect CNS function. Here we report the detection of early physiological abnormalities in simian immunodeficiency virus-infected monkeys. The acute infection caused a disruption of the circadian rhythm manifested by rises in body temperature, observed in all five individuals between 1 and 2 weeks postinoculation (p.i.), accompanied by a reduction in daily motor activity to 50% of control levels. Animals remained hyperthermic at 1 and 2 months p.i. and returned to preinoculation temperatures at 3 months after viral inoculation. Although motor activity recovered to baseline values at 1 month p.i., activity levels then decreased to approximately 50% of preinoculation values over the next 2 months. Analysis of sensory-evoked responses 1 month p.i. revealed distinct infection-induced changes in auditory-evoked potential peak latencies that persisted at 3 months after viral inoculation. These early physiological abnormalities may precede the development of observable cognitive or motor deficiencies and can provide an assay to evaluate agents to prevent or alleviate neuronal dysfunction.
The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42
Authors: Ma Le , Rohatgi Rajat , Kirschner Marc W.
Published: July 2002

The small GTP-binding protein Cdc42 is thought to induce filopodium formation by regulating actin polymerization at the cell cortex. Although several Cdc42-binding proteins have been identified and some of them have been implicated in filopodium formation, the precise role of Cdc42 in modulating actin polymerization has not been defined. To understand the biochemical pathways that link Cdc42 to the actin cytoskeleton, we have reconstituted Cdc42-induced actin polymerization in
Xenopus
egg extracts. Using this cell-free system, we have developed a rapid and specific assay that has allowed us to fractionate the extract and isolate factors involved in this activity. We report here that at least two biochemically distinct components are required, based on their chromatographic behavior and affinity for Cdc42. One component is purified to homogeneity and is identified as the Arp2/3 complex, a protein complex that has been shown to nucleate actin polymerization. However, the purified complex alone is not sufficient to mediate the activity; a second component that binds Cdc42 directly and mediates the interaction between Cdc42 and the complex also is required. These results establish an important link between a signaling molecule, Cdc42, and a complex that can directly modulate actin networks
in vitro
. We propose that activation of the Arp2/3 complex by Cdc42 and other signaling molecules plays a central role in stimulating actin polymerization at the cell surface.
Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) protect hippocampal neurons against excitatory amino acid-induced neurotoxicity
Authors: Kimonides V. G. , Khatibi N. H. , Svendsen C. N. , Sofroniew M. V. , Herbert J.
Published: July 2002

DHEA, together with DHEAS, is the most abundant steroid in the blood of young adult humans. Levels in humans decline with age and during certain types of illness or stress. We have found that DHEA(S) can prevent or reduce the neurotoxic actions in the hippocampus of the glutamate agonists
N
-methyl-
d
-aspartic acid (NMDA) both
in vitro
and
in vivo
or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid
in vitro
. Pre-treatment with DHEA (10–100 nM for 6–8 h) protected primary hippocampal cultures from embryonic day 18 (E18) embryos against NMDA-induced toxicity (0.1, 1, 10, and 50 mM). DHEA added either with NMDA (1 mM) or 1 h later had lesser, but still significant, protective actions. DHEAS also reduced NMDA-induced toxicity (1 mM), although the lowest effective dose of DHEAS (100 nM) was higher than that of DHEA (10 nM). DHEA (100 nM) protected cultured neurons against the neurotoxic actions of either AMPA (25 μM) or kainic acid (1 mM) as well.
In vivo
, s.c. pellets of DHEA, which resulted in plasma levels that resembled those in young adult humans, protected hippocampal CA1/2 neurons against unilateral infusions of 5 or 10 nmol of NMDA. Because the release of glutamate has been implicated in the neural damage after cerebral ischemia and other neural insults, these results suggest that decreased DHEA levels may contribute significantly to the increased vulnerability of the aging or stressed human brain to such damage.
Defining therapeutic targets by using adenovirus: Blocking NF-κB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators
Authors: Bondeson Jan , Foxwell Brian , Brennan Fionula , Feldmann Marc
Published: July 2002

The role of the transcription factor NF-κB in the pathogenesis of rheumatoid arthritis has long been a subject of controversy. We used an adenoviral technique of blocking NF-κB through overexpression of the inhibitory subunit IκBα, which has the advantage that it can be used in the diseased tissue itself, with >90% of the synovial macrophages, fibroblasts, and T cells infected. We found that the spontaneous production of tumor necrosis factor α and other pro-inflammatory cytokines is NF-κB-dependent in rheumatoid synovial tissue, in contrast to the main anti-inflammatory mediators, like IL-10 and -11, and the IL-1 receptor antagonist. Of even more interest, IκBα overexpression inhibited the production of matrix metalloproteinases 1 and 3 while not affecting their tissue inhibitor. Blocking NF-κB in the rheumatoid joint thus has a very beneficial profile, reducing both the inflammatory response and the tissue destruction. The adenoviral technique described here has widespread applicability, allowing rapid testing of the effects of blocking a potential therapeutic target in either cultures of normal cells or in the diseased tissue itself.
Distribution of tactile learning and its neural basis
Authors: Harris Justin A. , Petersen Rasmus S. , Diamond Mathew E.
Published: July 2002

The brain’s sensory processing systems are modified during perceptual learning. To learn more about the spatial organization of learning-related modifications, we trained rats to utilize the sensory signal from a single intact whisker to carry out a behavioral task. Once a rat had mastered the task, we clipped its “trained” whisker and attached a “prosthetic” one to a different whisker stub. We then tested the rat to determine how quickly it could relearn the task by using the new whisker. We observed that rats were immediately able to use the prosthetic whisker if it were attached to the stub of the trained whisker but not if it were attached to a different stub. Indeed, the greater the distance between the trained and prosthetic whisker, the more trials were needed to relearn the task. We hypothesized that this “transfer” of learning between whiskers might depend on how much the representations of individual whiskers overlap in primary somatosensory cortex. Testing this hypothesis by using 100-electrode cortical recordings, we found that the overlap between the cortical response patterns of two whiskers accounted well for the transfer of learning between them: The correlation between the electrophysiological and behavioral data was very high (
r
= 0.98). These findings suggest that a topographically distributed memory trace for sensory-perceptual learning may reside in primary sensory cortex.
A theoretical search for folding/unfolding nuclei in three-dimensional protein structures
Authors: Galzitskaya Oxana V. , Finkelstein Alexei V.
Published: July 2002

When a protein folds or unfolds, it has to pass through many half-folded microstates. Only a few of them can be seen experimentally. In a two-state transition proceeding with no accumulation of metastable intermediates [Fersht, A. R. (1995)Curr. Opin. Struct. Biol.5, 79–84], only the semifolded microstates corresponding to the transition state can be outlined; they influence the folding/unfolding kinetics. Our aim is to calculate them, provided the three-dimensional protein structure is given. The presented approach follows from the capillarity theory of protein folding and unfolding [Wolynes, P. G. (1997)Proc. Natl. Acad. Sci. USA94, 6170–6175]. The approach is based on a search for free-energy saddle point(s) on a network of protein unfolding pathways. Under some approximations, this search is rapidly performed by dynamic programming and, despite its relative simplicity, gives a good correlation with experiment. The computed folding nuclei look like ensembles of those compact and closely packed parts of the three-dimensional native folds that contain a small number of disordered protruding loops. Their estimated free energy is consistent with the rapid (within seconds) folding and unfolding of small proteins at the point of thermodynamic equilibrium between the native fold and the coil.
Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells
Authors: Kazantsev A. , Preisinger E. , Dranovsky A. , Goldgaber D. , Housman D.
Published: July 2002

Pathological degeneration of neurons in Huntington’s disease and associated neurodegenerative disorders is directly correlated with the expansion of CAG repeats encoding polyglutamines of extended length. The physical properties of extended polyglutamines and the intracellular consequences of expression of polyglutamine expansion have been the object of intensive investigation. We have extended the range of lengths of polyglutamine produced by recombinant DNA methodology by constructing a library of CAG/CAA repeats coding for a range of 25–300 glutamine residues. We have investigated the subcellular localization, interaction with other polyglutamine-containing polypeptides, and the physical properties of aggregated forms of polyglutamine in the cell. Extended polyQ aggregated in the cytoplasm and was only transported to the nucleus when a strong nuclear localization signal was present. Polyglutamine below pathological lengths could be captured in aggregates and transported to ectopic cell locations. The CREB-binding protein (CBP), containing a homopolymeric stretch of 19 glutamines, was likewise found to coaggregate in a polyglutamine-dependent manner, suggesting that pathology in polyglutamine disease may result from cellular depletion of normal proteins containing polyglutamine. We have observed a striking detergent resistance in aggregates produced from polyglutamine of pathological length. This observation has led to the development of a fluorescence-based assay exploiting the detergent resistance of polyglutamine aggregates that should facilitate high-throughput screening for agents that suppress polyglutamine aggregation in cells.
Fgfr2
is required for limb outgrowth and lung-branching morphogenesis
Authors: Arman Esther , Haffner-Krausz Rebecca , Gorivodsky Marat , Lonai Peter
Published: July 2002

The aim of this study was to clarify the role of
Fgfr2
during later stages of embryonic development. Of two previously reported gene-targeting experiments, the more extensive
Fgfr2
deletion was lethal shortly after implantation, because of trophoblast defects, whereas the less extensive one survived until midgestation with placental insufficiency and defective limb outgrowth [Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P. & Deng, C. (1998)
Development (Cambridge, U.K.)
125, 753–765].
Fgfr2
in the early embryo is expressed in the trophectoderm, and this extra-embryonic localization persists into mid- and late gestation, when
Fgfr2
also is expressed in multiple developing organs. To gain insight into the later functions of
Fgfr2,
fusion chimeras were constructed from homozygous mutant embryonic stem cells and wild-type tetraploid embryos. This allowed survival until term and revealed that
Fgfr2
is required for both limb outgrowth and branching lung morphogenesis. The use of fusion chimeras demonstrated that early lethality was indeed because of trophectoderm defects and indicated that in the embryonic cell lineages
Fgfr2
activity manifests in limb and lung development. Highly similar lung and limb phenotypes were detected recently in the loss of function mutation of
Fgf10,
a ligand of
Fgfr2
. It is likely, therefore, that whereas during early development
Fgfr2
interacts with
Fgf4
, in limb and lung development interactions between
Fgf10
and
Fgfr2
may be required. Possible epithelial–mesenchymal interactions between the splicing alternatives of
Fgfr2
and their specific ligands will be discussed.
IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils
Authors: Yoshimoto Tomohiro , Tsutsui Hiroko , Tominaga Kouji , Hoshino Katsuaki , Okamura Haruki , Akira Shizuo , Paul William E. , Nakanishi Kenji
Published: July 2002

IL-18 is a proinflammatory cytokine that plays an important role in natural killer cell activation and T helper 1 (Th1) cell responses. Mast cells and basophils are major inducers and effectors of allergic inflammation. Here we show that basophils and mast cells derived by culture of bone marrow cells with IL-3 for 10 days express IL-18Rα chain and that basophils produce large amounts of IL-4 and IL-13 in response to stimulation with IL-3 and IL-18. Injection of IL-12 and IL-18 inhibits IgE production in helminth-infected wild-type mice and abolishes the capacity of their basophils to produce IL-4 and IL-13 in response to stimulation either with IL-3 and IL-18 or with FcɛR cross-linkage. By contrast, this combination of cytokines actually increases IgE levels in helminth-infected IFN-γ−/−mice and enhances IL-4 and IL-13 production by their basophils. Furthermore, injection of IL-18 alone enhances basophil production of IL-4 and histamine both in wild-type and IFN-γ−/−mice. Thus, IL-18 has the potential to stimulate basophils but, when given with IL-12, exhibits an antiallergic actionin vivo.
Two serine residues of the glutamate transporter GLT-1 are crucial for coupling the fluxes of sodium and the neurotransmitter
Authors: Zhang Yumin , Kanner Baruch I.
Published: July 2002

The neurotoxicity of glutamate in the central nervous system is
restricted by several (Na
+
+ K
+
)-coupled
transporters for this neurotransmitter. The astroglial transporter
GLT-1 is the only subtype that exhibits high sensitivity to the
nontransportable glutamate analogue dihydrokainate. A marked reduction
in sensitivity to the blocker is observed when serine residues 440 and
443 are mutated to glycine and glutamine, which, respectively, occupy
these positions in the other homologous glutamate transporters. They
are located in the ascending limb of the recently identified
pore-loop-like structure. Strikingly, mutation of serine-440 to glycine
enables not only sodium but also lithium ions to drive net influx of
acidic amino acids. Moreover, the efficiency of lithium as a driving
ion for glutamate transport depends on the nature of the amino acid
residue present at position 443. Mutant transporters containing single
cysteines at the position of either serine residue become sensitive to
positively as well as negatively charged methanethiosulfonate
derivatives. In S440C transporters significant protection against this
inhibition is provided both by transportable and nontransportable
glutamate analogues, but not by sodium alone. Our observations indicate
that the pore-loop-like structure plays a pivotal role in coupling ion
and glutamate fluxes and suggest that it is close to the
glutamate-binding site.
A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs
Authors: Reid Carl E. , Lazinski David W.
Published: July 2002

Hepatitis δ virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5′OH and 2′,3′-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5′ hydroxyl and 2′,3′- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not
in vitro
or in
Escherichia coli
. The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus.
The preaggregated state of an amyloidogenic protein: Hydrostatic pressure converts native transthyretin into the amyloidogenic state
Authors: Ferrão-Gonzales Astria D. , Souto Sandro O. , Silva Jerson L. , Foguel Débora
Published: July 2002

Protein misfolding and aggregation cause several diseases, by mechanisms that are poorly understood. The formation of amyloid aggregates is the hallmark of most of these diseases. Here, the properties and formation of amyloidogenic intermediates of transthyretin (TTR) were investigated by the use of hydrostatic pressure and spectroscopic techniques. Native TTR tetramers (T
4
) were denatured by high pressure into a conformation that exposes tryptophan residues to the aqueous environment. This conformation was able to bind the hydrophobic probe bis-(8-anilinonaphthalene-1-sulfonate), indicating persistence of elements of secondary and tertiary structure. Lowering the temperature facilitated the pressure-induced denaturation of TTR, which suggests an important role of entropy in stabilizing the native protein. Gel filtration chromatography showed that after a cycle of compression-decompression at 1°C, the main species present was a tetramer, with a small population of monomers. This tetramer, designated T
4
*, had a non-native conformation: it bound more bis-(8-anilinonaphthalene-1-sulfonate) than native T
4
, was less stable under pressure, and on decompression formed aggregates under mild acidic conditions (pH 5–5.6). Our data show that hydrostatic pressure converts native tetramers of TTR into an altered state that shares properties with a previously described amyloidogenic intermediate, and it may be an intermediate that lies on the aggregation pathway. This “preaggregated” state, which we call T
4
*, provides insight into the question of how a correctly folded protein may degenerate into the aggregation pathway in amyloidogenic diseases.
Anthropoid humeri from the late Eocene of Egypt
Authors: Seiffert Erik R. , Simons Elwyn L. , Fleagle John G.
Published: July 2002

A number of recent studies have, by necessity, placed a great deal of emphasis on the dental evidence for Paleogene anthropoid interrelationships, but cladistic analyses of these data have led to the erection of phylogenetic hypotheses that appear to be at odds with biogeographic and stratigraphic considerations. Additional morphological data from the cranium and postcranium of certain poorly understood Paleogene primates are clearly needed to help test whether such hypotheses are tenable. Here we describe humeri attributable toProteopithecus sylviaeandCatopithecus browni, two anthropoids from late Eocene sediments of the Fayum Depression in Egypt. Qualitative and morphometric analyses of these elements indicate that humeri of the oligopithecineCatopithecusare more similar to early Oligocene propliopithecines than they are to any other Paleogene anthropoid taxon, and thatProteopithecusexhibits humeral similarities to parapithecids that may be symplesiomorphies of extant (or “crown”) Anthropoidea. The humeral morphology ofCatopithecusis consistent with certain narrowly distributed dental apomorphies—such as the loss of the upper and lower second premolar and the development of a honing blade for the upper canine on the lower third premolar—which suggest that oligopithecines constitute the sister group of a clade containing propliopithecines and Miocene-Recent catarrhines and are not most closely related toProteopithecusas has recently been proposed.
Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype
Authors: Toyota Minoru , Ohe-Toyota Mutsumi , Ahuja Nita , Issa Jean-Pierre J.
Published: July 2002

Colorectal cancers (CRCs) are characterized by multiple genetic (mutations) and epigenetic (CpG island methylation) alterations, but it is not known whether these evolve independently through stochastic processes. We have recently described a novel pathway termed CpG island methylator phenotype (CIMP) in CRC, which is characterized by the simultaneous methylation of multiple CpG islands, including several known genes, such as
p16
,
hMLH1
, and
THBS1
. We have now studied mutations in
K-RAS
,
p53
,
DPC4
, and
TGFβRII
in a panel of colorectal tumors with or without CIMP. We find that CIMP defines two groups of tumors with significantly different genetic lesions: frequent
K-RAS
mutations were found in CIMP
+
CRCs (28/41, 68%) compared with CIMP
−
cases (14/47, 30%,
P
= 0.0005). By contrast,
p53
mutations were found in 24% (10/41) of CIMP
+
CRCs vs. 60% (30/46) of CIMP
−
cases (
P
= 0.002). Both of these differences were independent of microsatellite instability. These interactions between CIMP,
K-RAS
mutations, and
p53
mutations were preserved in colorectal adenomas, suggesting that they occur early in carcinogenesis. The distinct combinations of epigenetic and genetic alterations in each group suggest that activation of oncogenes and inactivation of tumor suppressor genes is related to the underlying mechanism of generating molecular diversity in cancer, rather than simply accumulate stochastically during cancer development.
Is war a good or a bad thing? The attitudes of Croatian, Israeli, and Palestinian children toward war
Authors: Jagodić Gordana Kuterovac
Published: July 2002

It has been documented that children's basic attitudes to social issues such as war and peace are formed during the early school‐age period. The aims of the study were threefold: (1) to examine the attitudes of Croatian school‐age children toward war and peace; (2) to explore the relationship between these attitudes and children's gender, age, and amount of experience with war and violence; and (3) to compare the attitudes of Croatian children toward war and peace with the attitudes of Israeli and Palestinian children examined using the same scale 18 years earlier. The sample consisted of 230 children aged 11–14 years who attended school in Zagreb. The results indicate that Croatian children generally have a negative attitude toward war, but strongly support the fight for the freedom of their nation. A relationship was found between the children's gender, age, and war experiences and their attitudes toward war. A comparison between Croatian, Israeli, and Palestinian children showed that nationhood had an influence on attitudes toward war. However, there is greater similarity in the attitudes of today's Croatian and former Israeli children than between today's Croatian and former Palestinian children, and between Israeli and Palestinian children 18 years ago.
Friendships in Early Childhood, and Social Understanding
Authors: Maguire Mary C. , Dunn Judy
Published: July 2002

This study had two goals. The first was to provide descriptive data on the nature of individual differences in young children’s close friendships, and the second, to examine the relations between these individual differences and children’s earlier understanding of others’ emotions and mental states, and their later appreciation of ambivalent or mixed emotions. A total of 41 children participating in a longitudinal study from 33 months to 6-7 years were studied with their close friends as 6-year-olds, with a combination of observations and standard sociocognitive assessments. The results showed that different aspects of friendship interactions, such as co-ordination of play and amity, were neither closely related nor linked to power assertion. Early differences in the assessment of social understanding were associated with later differences in pretend play with the friend, and friendship interactions at 6 years were linked to later appreciation of mixed emotions. The two-way process of influence linking individual development and friendship quality is discussed.
Modelling Synergy using Manifest Categorical Variables
Authors: von Eye Alexander , Schuster Christof , Rogers William M.
Published: July 2002

This paper discusses methods to model the concept of synergy at the level of manifest categorical variables. First, a classification of concepts of synergy is presented. A dditive and nonadditive concepts of synergy are distinguished. Most prominent among the nonadditive concepts is superadditive synergy. Examples are given from the natural sciences and the social sciences. M delling focuses on the relationship between the agents involved in a synergetic process. These relationships are expressed in form of contrasts, expressed in effect coding vectors in design matrices for nonstandard log-linear models. A method by Schuster is used to transform design matrices such that parameters reflect the proposed relationships. A n example reanalyses data presented by Bishop, Fienberg, and Holland (1975) that describe the development of thromboembolisms in women who differ in their patterns of contraceptive use and smoking. Alternative methods of analysis are com pared. Implications for developmental research are discussed.
The Pharmacokinetics of Methadone Following Co‐Administration with a Lamivudine/Zidovudine Combination Tablet in Opiate‐Dependent Subjects
Authors: Rainey Petrie M. , Friedland Gerald H. , Snidow Jerry W. , McCance‐Katz Elinore F. , Mitchell Susan M. , Andrews Laurie , Lane Barbara , Jatlow Peter
Published: July 2002

Methadone pharmacokinetics were determined in an open‐label, within subject study in 16 methadone‐maintained, non‐HIV‐infected subjects prior to and following administration of one lamivudine 150‐mg/zidovudine 300‐mgcombi‐nation tablet to determine whether this antiretroviral therapy alters methadone serum concentrations. No significant differences in the mean are aunder the serum concentration‐time curve (AUC0‐24h; 8753 ± 4280 vs. 8641 ± 4431 μg‐h/L), oralclearance(CL/F;9.9± 4.9vs.10.3 ± 5.5 L/h), oral volume of distribution (Vd/F; 647 ± 465 vs. 481 ± 305 L), maximum serum concentration (Cmax; 514 ± 223 vs. 5510 ± 237 μg/L), or terminal elimination half‐life (tl/2; 55.3 ± 61.0 vs. 35.0 ± 17.5 h) were detected. These results suggest that methadone dose change is not likely to be necessary for patients treated with lamivudine/zidovudine combination pharmacotherapy.
Expression of Smooth Muscle Calponin in Synovial Sarcoma
Authors: Ono Hidefumi , Yoshikawa Hideki , Ueda Takafumi , Yamamura Hisako , Kudawara Ikuo , Manou Masayuki , Ishiguro Shingo , Funai Hiroko , Koyanagi Yumiko , Araki Nobuhito , Hashimoto Nobuyuki , Sonobe Hiroshi , Tatsuta Masaharu , Takahashi Katsuhito
Published: July 2002

Purpose. Histogenesis of synovial sarcoma remains controversial
and reliable molecular markers for diagnosis are necessary. Expression of basic calponin,
a smooth muscle differentiation‐specific actin‐binding protein, was studied in
synovial sarcoma.

Subjects and Methods. The basic calponin gene and the gene
product were analyzed by reverse transcription PCR analysis (RT‐PCR) and
immunohistochemistry in 14 synovial sarcomas and a human synovial sarcoma
cell line (HS‐SY‐II).

Results and Discussion. Immunoreactivity for basic calponin
was detected in the cytoplasm of 6 synovial sarcomas (43% positive). In the basic
calponin‐positive tumors and the HS‐SY‐II cells, expression for smooth muscle‐specific
genes, including basic calponin and SM22α , was detected by RT‐PCR, suggesting a
lineage relationship between synovial sarcoma cells and smooth muscle‐like
mesenchymal cells.

Conclusions. A subset of synovial sarcomas expressing the basic
calponin gene and the gene product were identified. The basic calponin may have
potential utility as a novel molecular marker identifying certain synovial sarcomas.
Pitch Change In Male‐To‐Female Transsexuals: Has Phonosurgery A Role To Play?
Authors: Brown M. , Perry A. , Cheesman A. D. , Pring T.
Published: July 2002

Male‐to‐female transsexuals, who have undergone gender reassignment surgery, may continue to have low pitched voices. Voice therapy may assist them to use their voice in a manner more likely to be perceived as female but, if this approach is unsuccessful, a laryngeal framework operation may be necessary to raise vocal pitch. This study assessed the effects of crico‐thyroid approximation surgery in 14 transsexuals. Modal pitch was significantly increased by surgery but with substantial variation across speakers. Modal pitch was significantly correlated with judgements of gender by speech and language therapists who listened to tapes of the subjects. The results suggest that crico‐thyroid approximation may be used to raise the pitch of voices of male‐to‐female transsexuals. Further research is needed to clarify the reasons for the variability in outcome, to monitor the longer‐term changes in voice and the impact for clients of their modified voice in real life situations.
Chromosomal Proteins and Cytokinesis: Patterns of Cleavage Furrow Formation and Inner Centromere Protein Positioning in Mitotic Heterokaryons and Mid-anaphase Cells
Authors: Eckley D. Mark , Ainsztein Alexandra M. , Mackay Alastair M. , Goldberg Ilya G. , Earnshaw William C.
Published: July 2002

After the separation of sister chromatids in anaphase, it is essential that the cell position a cleavage furrow so that it partitions the chromatids into two daughter cells of roughly equal size. The mechanism by which cells position this cleavage furrow remains unknown, although the best current model is that furrows always assemble midway between asters. We used micromanipulation of human cultured cells to produce mitotic heterokaryons with two spindles fused in a V conformation. The majority (15/19) of these cells cleaved along a single plane that transected the two arms of the V at the position where the metaphase plate had been, a result at odds with current views of furrow positioning. However, four cells did form an additional ectopic furrow between the spindle poles at the open end of the V, consistent with the established view. To begin to address the mechanism of furrow assembly, we have begun a detailed study of the properties of the chromosome passenger inner centromere protein (INCENP) in anaphase and telophase cells. We found that INCENP is a very early component of the cleavage furrow, accumulating at the equatorial cortex before any noticeable cortical shape change and before any local accumulation of myosin heavy chain. In mitotic heterokaryons, INCENP was detected in association with spindle midzone microtubules beneath sites of furrowing and was not detected when furrows were absent. A functional role for INCENP in cytokinesis was suggested in experiments where a nearly full-length INCENP was tethered to the centromere. Many cells expressing the chimeric INCENP failed to complete cytokinesis and entered the next cell cycle with daughter cells connected by a large intercellular bridge with a prominent midbody. Together, these results suggest that INCENP has a role in either the assembly or function of the cleavage furrow.