bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell surface expression of the Alzheimer disease-related presenilin proteins
Authors: Dewji Nazneen N. , Singer S. J.
Published: July 2002

The presenilin proteins PS-1 and PS-2 are crucially involved in Alzheimer disease (AD), but their molecular functions are not known. They are integral membrane proteins, but whether they can be expressed at the surface of cells has been in dispute. Here we show by immunofluorescence experiments, using anti-peptide antibodies specific for either PS-1 or PS-2, that live cultured DAMI cells and differentiated human NT2N neuronal cells are specifically immunolabeled for their endogenous as well as transfected presenilins, although the cells cannot be immunolabeled for their intracellular tubulin, unless they are first fixed and permeabilized. These and other results establish that portions of the presenilins are indeed expressed at the surfaces of these cells. These findings support our previous proposal that the presenilins on the surface of a cell engage in intercellular interactions with the β-amyloid precursor protein on the surface of a neighboring cell, as a critical step in the molecular and cellular mechanisms that lead to AD.
Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression
Authors: Qing Keyun , Wang Xu-Shan , Kube Dagmar M. , Ponnazhagan Selvarangan , Bajpai Anil , Srivastava Arun
Published: July 2002

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3′ end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.
Neurosteroids: Deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus
Authors: Vallée Monique , Mayo Willy , Darnaudéry Muriel , Corpéchot Colette , Young Jacques , Koehl Muriel , Le Moal Michel , Baulieu Etienne-Emile , Robel Paul , Simon Hervé
Published: July 2002

Pregnenolone sulfate (PREG S) is synthesized in the nervous system and is a major neurosteroid in the rat brain. Its concentrations were measured in the hippocampus and other brain areas of single adult and aged (22–24 month-old) male Sprague–Dawley rats. Significantly lower levels were found in aged rats, although the values were widely scattered and reached, in about half the animals, the same range as those of young ones. The spatial memory performances of aged rats were investigated in two different spatial memory tasks, the Morris water maze and Y-maze. Performances in both tests were significantly correlated and, accompanied by appropriate controls, likely evaluated genuine memory function. Importantly, individual hippocampal PREG S and distance to reach the platform in the water maze were linked by a significant correlation, i.e., those rats with lower memory deficit had the highest PREG S levels, whereas no relationship was found with the PREG S content in other brain areas (amygdala, prefrontal cortex, parietal cortex, striatum). Moreover, the memory deficit of cognitively impaired aged rats was transiently corrected after either intraperitoneal or bilateral intrahippocampal injection of PREG S. PREG S is both a γ-aminobutyric acid antagonist and a positive allosteric modulator at the
N
-methyl-
d
-aspartate receptor, and may reinforce neurotransmitter system(s) that decline with age. Indeed, intracerebroventricular injection of PREG S was shown to stimulate acetylcholine release in the adult rat hippocampus. In conclusion, it is proposed that the hippocampal content of PREG S plays a physiological role in preserving and/or enhancing cognitive abilities in old animals, possibly via an interaction with central cholinergic systems. Thus, neurosteroids should be further studied in the context of prevention and/or treatment of age-related memory disorders.
Predominance of the α
1D
subunit in L-type voltage-gated Ca
2+
channels of hair cells in the chicken’s cochlea
Authors: Kollmar Richard , Montgomery Lisa G. , Fak John , Henry Lisa J. , Hudspeth A. J.
Published: July 2002

The voltage-gated Ca
2+
channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca
2+
channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca
2+
channels, we investigated the expression of L-type α
1
subunits in hair cells of the chicken’s cochlea. In PCRs with five different pairs of degenerate primers, we always obtained α
1D
products, but only once an α
1C
product and never an α
1S
product. A full-length α
1D
mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the α
1D
subunit was about 90% identical to those of the mammalian α
1D
subunits.
In situ
hybridization confirmed that the α
1D
mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the α
1D
mRNA is 100–500 times more abundant than the α
1C
mRNA. We conclude that most, if not all, voltage-gated Ca
2+
channels in hair cells contain an α
1D
subunit. Furthermore, we propose that the α
1D
subunit plays a hitherto undocumented role at tonic synapses.
Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in
Saccharomyces cerevisiae
Authors: Ludin Katja , Jiang Rong , Carlson Marian
Published: July 2002

The Snf1 protein kinase family has been conserved in eukaryotes. In the yeast
Saccharomyces cerevisiae
, Snf1 is essential for transcription of glucose-repressed genes in response to glucose starvation. The direct interaction between Snf1 and its activating subunit, Snf4, within the kinase complex is regulated by the glucose signal. Glucose inhibition of the Snf1-Snf4 interaction depends on protein phosphatase 1 and its targeting subunit, Reg1. Here we show that Reg1 interacts with the Snf1 catalytic domain in the two-hybrid system. This interaction increases in response to glucose limitation and requires the conserved threonine in the activation loop of the kinase, a putative phosphorylation site. The inhibitory effect of Reg1 appears to require the Snf1 regulatory domain because a
reg1Δ
mutation no longer relieves glucose repression of transcription when Snf1 function is provided by the isolated catalytic domain. Finally, we show that abolishing the Snf1 catalytic activity by mutation of the ATP-binding site causes elevated, constitutive interaction with Reg1, indicating that Snf1 negatively regulates its own interaction with Reg1. We propose a model in which protein phosphatase 1, targeted by Reg1, facilitates the conformational change of the kinase complex from its active state to the autoinhibited state.
Protein kinase CK2 interacts with and phosphorylates the
Arabidopsis
circadian clock-associated 1 protein
Authors: Sugano Shoji , Andronis Christos , Green Rachel M. , Wang Zhi-Yong , Tobin Elaine M.
Published: July 2002

The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of
Lhcb1*3
gene expression and in the function of the circadian oscillator in
Arabidopsis thaliana
. By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (β) subunit of the protein kinase CK2 and have designated it as
CKB3
.
CKB3
is the only reported example of a third β-subunit of CK2 found in any organism. CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an
in vitro
interaction assay. Other subunits of CK2 also show an interaction with CCA1
in vitro
. CK2 β-subunits stimulate binding of CCA1 to the CCA1 binding site on the
Lhcb1*3
gene promoter, and recombinant CK2 is able to phosphorylate CCA1
in vitro
. Furthermore,
Arabidopsis
plant extracts contain a CK2-like activity that affects the formation of a DNA–protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1
in vivo
.
Eukaryotic phytochromes: Light-regulated serine/threonine protein kinases with histidine kinase ancestry
Authors: Yeh Kuo-Chen , Lagarias J. Clark
Published: July 2002

The discovery of cyanobacterial phytochrome histidine kinases, together with the evidence that phytochromes from higher plants display protein kinase activity, bind ATP analogs, and possess C-terminal domains similar to bacterial histidine kinases, has fueled the controversial hypothesis that the eukaryotic phytochrome family of photoreceptors are light-regulated enzymes. Here we demonstrate that purified recombinant phytochromes from a higher plant and a green alga exhibit serine/threonine kinase activity similar to that of phytochrome isolated from dark grown seedlings. Phosphorylation of recombinant oat phytochrome is a light- and chromophore-regulated intramolecular process. Based on comparative protein sequence alignments and biochemical cross-talk experiments with the response regulator substrate of the cyanobacterial phytochrome Cph1, we propose that eukaryotic phytochromes are histidine kinase paralogs with serine/threonine specificity whose enzymatic activity diverged from that of a prokaryotic ancestor after duplication of the transmitter module.
Hippocampal, but not amygdala, activity at encoding correlates with long-term, free recall of nonemotional information
Authors: Alkire Michael T. , Haier Richard J. , Fallon James H. , Cahill Larry
Published: July 2002

Participation of two medial temporal lobe structures, the hippocampal region and the amygdala, in long-term declarative memory encoding was examined by using positron emission tomography of regional cerebral glucose. Positron emission tomography scanning was performed in eight healthy subjects listening passively to a repeated sequence of unrelated words. Memory for the words was assessed 24 hr later with an incidental free recall test. The percentage of words freely recalled then was correlated with glucose activity during encoding. The results revealed a striking correlation (
r
= 0.91,
P
< 0.001) between activity of the left hippocampal region (centered on the dorsal parahippocampal gyrus) and word recall. No correlation was found between activity of either the left or right amygdala and recall. The findings provide evidence for hippocampal involvement in long-term declarative memory encoding and for the view that the amygdala is not involved with declarative memory formation for nonemotional material.
Mice with type 2 and 3 Gaucher disease point mutations generated by a single insertion mutagenesis procedure (SIMP)
Authors: Liu Yujing , Suzuki Kinuko , Reed Jennifer D. , Grinberg Alexander , Westphal Heiner , Hoffmann Alexander , Döring Thomas , Sandhoff Konrad , Proia Richard L.
Published: July 2002

Gaucher disease is caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC). Three clinical types of Gaucher disease have been defined according to the presence (type 2 and 3) or absence (type 1) of central nervous system disease and severity of clinical manifestations. The clinical course of the disease correlates with the mutation carried by the GC gene. To produce mice with point mutations that correspond to the clinical types of Gaucher disease, we have devised a highly efficient one-step mutagenesis method—the single insertion mutagenesis procedure (SIMP)—to introduce human disease mutations into the mouse GC gene. By using SIMP, mice were generated carrying either the very severe RecNciI mutation that can cause type 2 disease or the less severe L444P mutation associated with type 3 disease. Mice homozygous for the RecNciI mutation had little GC enzyme activity and accumulated glucosylceramide in brain and liver. In contrast, the mice homozygous for the L444P mutation had higher levels of GC activity and no detectable accumulation of glucosylceramide in brain and liver. Surprisingly, both point mutation mice died within 48 hr of birth, apparently of a compromised epidermal permeability barrier caused by defective glucosylceramide metabolism in the epidermis.
Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin
Authors: Jabaudon D. , Shimamoto K. , Yasuda-Kamatani Y. , Scanziani M. , Gähwiler B. H. , Gerber U.
Published: July 2002

Maintaining glutamate at low extracellular concentrations in the central nervous system is necessary to protect neurons from excitotoxic injury and to ensure a high signal-to-noise ratio for glutamatergic synaptic transmission. We have used
d
l
-threo-β-benzyloxyaspartate (TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration. By using the
N
-methyl-
d
-aspartate receptors of patched CA3 hippocampal neurons as “glutamate sensors,” we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration. This increase was Ca
2+
-independent and was observed in the presence of tetrodotoxin. Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited. Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process. TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter. This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange. These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment. As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.
Developmental regulation of spine motility in the mammalian central nervous system
Authors: Dunaevsky Anna , Tashiro Ayumu , Majewska Ania , Mason Carol , Yuste Rafael
Published: July 2002

The function of dendritic spines, postsynaptic sites of excitatory input in the mammalian central nervous system (CNS), is still not well understood. Although changes in spine morphology may mediate synaptic plasticity, the extent of basal spine motility and its regulation and function remains controversial. We investigated spine motility in three principal neurons of the mouse CNS: cerebellar Purkinje cells, and cortical and hippocampal pyramidal neurons. Motility was assayed with time-lapse imaging by using two-photon microscopy of green fluorescent protein-labeled neurons in acute and cultured slices. In all three cell types, dendritic protrusions (filopodia and spines) were highly dynamic, exhibiting a diversity of morphological rearrangements over short (<1-min) time courses. The incidence of spine motility declined during postnatal maturation, but dynamic changes were still apparent in many spines in late-postnatal neurons. Although blockade or induction of neuronal activity did not affect spine motility, disruption of actin polymerization did. We hypothesize that this basal motility of dendritic protrusions is intrinsic to the neuron and underlies the heightened plasticity found in developing CNS.
Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators
Authors: Yan Dalai , Cho Ho S. , Hastings Curtis A. , Igo Michele M. , Lee Seok-Yong , Pelton Jeffrey G. , Stewart Valley , Wemmer David E. , Kustu Sydney
Published: July 2002

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.
Alzheimer-like neurodegeneration in aged antinerve growth factor transgenic mice
Authors: Capsoni Simona , Ugolini Gabriele , Comparini Alessandro , Ruberti Francesca , Berardi Nicoletta , Cattaneo Antonino
Published: July 2002

Neurotrophin nerve growth factor (NGF) has been suggested to be involved in age-related neurodegenerative diseases, but no transgenic model is currently available to study this concept. We have obtained transgenic mice expressing a neutralizing anti-NGF recombinant antibody, in which the levels of antibodies are three orders of magnitude higher in adult than in newborn mice [F.R., S. C., A.C., E. Di Daniel, J. Franzot, S. Gonfloni, G. Rossi, N. B. & A. C. (2000)
J. Neurosci.
, 20, 2589–2601]. In this paper, we analyze the phenotype of aged anti-NGF transgenic mice and demonstrate that these mice acquire an age-dependent neurodegenerative pathology including amyloid plaques, insoluble and hyperphosphorylated τ, and neurofibrillary tangles in cortical and hippocampal neurons. Aged anti-NGF mice also display extensive neuronal loss throughout the cortex, cholinergic deficit in the basal forebrain, and behavioral deficits. The overall picture is strikingly reminiscent of human Alzheimer's disease. Aged anti-NGF mice represent, to our knowledge, the most comprehensive animal model for this severe neurodegenerative disease. Also, these results demonstrate that, in mice, a deficit in the signaling and/or transport of NGF leads to neurodegeneration.
Modeling the fission yeast cell cycle: Quantized cycle times in
wee1
−
cdc25Δ
mutant cells
Authors: Sveiczer Akos , Csikasz-Nagy Attila , Gyorffy Bela , Tyson John J. , Novak Bela
Published: July 2002

A detailed mathematical model for the fission yeast mitotic cycle is developed based on positive and negative feedback loops by which Cdc13/Cdc2 kinase activates and inactivates itself. Positive feedbacks are created by Cdc13/Cdc2-dependent phosphorylation of specific substrates: inactivating its negative regulators (Rum1, Ste9 and Wee1/Mik1) and activating its positive regulator (Cdc25). A slow negative feedback loop is turned on during mitosis by activation of Slp1/anaphase-promoting complex (APC), which indirectly re-activates the negative regulators, leading to a drop in Cdc13/Cdc2 activity and exit from mitosis. The model explains how fission yeast cells can exit mitosis in the absence of Ste9 (Cdc13 degradation) and Rum1 (an inhibitor of Cdc13/Cdc2). We also show that, if the positive feedback loops accelerating the G
2
/M transition (through Wee1 and Cdc25) are weak, then cells can reset back to G
2
from early stages of mitosis by premature activation of the negative feedback loop. This resetting can happen more than once, resulting in a quantized distribution of cycle times, as observed experimentally in
wee1
−
cdc25Δ
mutant cells. Our quantitative description of these quantized cycles demonstrates the utility of mathematical modeling, because these cycles cannot be understood by intuitive arguments alone.
Dependence of lymphopenia-induced T cell proliferation on the abundance of peptide/ MHC epitopes and strength of their interaction with T cell receptors
Authors: Ge Qing , Rao Varada P. , Cho Bryan K. , Eisen Herman N. , Chen Jianzhu
Published: July 2002

Factors that affect naïve T cell proliferation in syngeneic lymphopenic hosts were investigated. 2C T cell receptor (TCR) transgenic T cells lacking both CD8 and CD4 survived but hardly proliferated. Proliferation of CD8+2C cells was proportional to the abundance of cognate peptide/MHC complexes and was severely inhibited by injection of anti-CD8 antibody. Weakly reactive self-peptides slightly enhanced CD8+2C cell proliferation whereas a potent agonist peptide promoted much more rapid proliferation, but inflammation-stimulating adjuvant had only a small effect on the rate of cell proliferation. The findings suggest that under uniform lymphopenic conditions, the widely different rates of proliferation of T cells expressing various TCR, or the same TCR in the presence or absence of CD8, reflect the strength of interaction between TCR and MHC associated with particular self-peptides.
Determinants of Short‐term Forgetting: Decay, Retroactive Interference, or Proactive Interference?
Authors: Tolan Georgina Anne , Tehan Gerald
Published: July 2002

In two experiments short‐term forgetting was investigated in a short‐term cued recall task designed to examine proactive interference effects. Mixed modality study lists were tested at varying retention intervals using verbal and nonverbal distractor activities. When an interfering foil was read aloud and a target item read silently, strong PI effects were observed for both types of distractor activity. When the target was read aloud and followed by a verbal distractor activity, weak PI effects emerged. However, when a target item was read aloud and nonverbal distractor activity filled the retention interval, performance was immune to the effects of PI for at least 8 seconds. The results indicate that phonological representations of items read aloud still influence performance after 15 seconds of distractor activity.
Relationships between eye contact, maternal sensitivity, and infant crying
Authors: Lohaus Arnold , Keller Heidi , Voelker Susanne
Published: July 2002

This study addresses the longitudinal association between eye contact and maternal sensitivity during the ” rst 12 weeks of the infants’ life and relates both variables to the infants’ cry behaviour at three months of age. Free-play interactional sequences of 20 mothers were videotaped at weekly intervals. At the infants’ age of three months, the mothers were additionally asked to record their infants’ cry behaviour using a maternal diary method over a period of three days. The amount of mutual eye contact was assessed microanalytically, and rating procedures were used to assess maternal sensitivity. Duration and frequency of infant crying were taken from the maternal diaries. The results show an increase of eye contact during the ” rst three months, and maternal sensitivity is a stable feature remaining almost unchanged over time. This difference may explain why early sensitivity ratings cannot predict later eye contact rates, whereas, in contrast, early eye contact measures are significantly related to later maternal sensitivity. In addition, the results show that the eye contact rate is (in contrast to maternal sensitivity) related to a decrease in the duration of infants’ crying.
Distinctions between Implicit and Explicit Memory: Significance for Understanding Cognitive Development
Authors: Anooshian Linda J.
Published: July 2002

This research was designed to explore developmental trends for implicit and explicit memory as well as relations between memory measures and other aspects of cognitive development for preschool and second-grade children, and adults. For one task, children and adults gradually clarified patterns of random dots until they could identify an emerging picture, in each of two testing sessions separated by one week. Reliable effects for testing session and age group were obtained for explicit memory (verbal recall as well as frequency and recognition ratings), but not for implicit memory. Typicality ratings, also obtained in this picture-clarification task, appeared more related to implicit than explicit memory. For other tasks, children and adults completed word stems (e.g. BR- - -) and provided examples of categories (e.g. animals). The absence of developmental differences for implicit memory contrasted with clear developmental improvement for recall (explicit memory). Other tasks—perceptual classification, and, for younger children, assessments of theories of mind—provided other measures of cognitive development. Measures of perceptual classification were generally unrelated to implicit or explicit memory; for preschoolers with poor explicit memory, naive theories of mind were associated with good implicit memory. Results were discussed primarily in terms of the significance of both implicit and explicit memory for understanding diverse areas of cognitive development.
Influence of Cognitive Development, Self-competency, and Teacher Evaluations on the Development of Children’s Racial Identity
Authors: Corenblum B. , Annis R. C. , Tanaka J. S.
Published: July 2002

Levels of cognitive development and perceived self-competencies have been shown to predict attitudes held by children in both minority and majority groups toward own-group members. Teacher appraisals may also influence children’s own-group attitudes by enforcing category-based expectancies and stereotypes about children’s group membership. To test this idea, White and Native Indian children in kindergarten, grades 1 and 2, answered recognition, similarity, and evaluation questions by pointing to pictures of Whites, Natives, and Blacks. Measures of children’s concrete operational thought and self-competency were obtained, as were classroom teacher ratings, of each child’s cognitive ability, peer acceptance, and physical development. Structural equation models indicated that teacher evaluations predicted White children’s, but not Native children’s own-group attitudes. Teacher ratings of Native children’s competencies did not predict minority children’s attitudes about themselves or own-group members. Implications of these findings for teacher expectancy effects and factors influencing teacher’s judgements of majority and minority group children were discussed.
Microgenetic Change in the Quantity and Quality of Preschoolers’ Private Speech
Authors: Duncan Robert M. , Pratt Michael W.
Published: July 2002

Forty preschool-aged children were videotaped while carrying out paper-folding and story-sequencing tasks, during a series of three experimental sessions. During the first session, participants worked on both easy and difficult items, and in the second and third sessions they worked on familiar items (the first session difficult items, presented repeatedly) and novel items, of each task type. Participants used more private speech on difficult/novel items than on easy/familiar items, during all three sessions. Private speech production declined across sessions when participants worked on the repeated items. A greater percentage of participants’ private speech preceded action when they worked on difficult/novel items, compared with easy/familiar items. On the paper-folding items, a cross-session increase occurred in the percentage of private speech that preceded action, supporting some of Vygotsky’s (1934/1987, 1978) claims about the emergence of verbal planning in private speech. The potential of microgenetic experimental methodology for research on private speech is emphasised.
Development of Reactions to Pain of Inoculation in Children and their Mothers
Authors: Negayama Koichi
Published: July 2002

This is a naturalistic study focusing on developmental changes in the immediate reactions of 183 Japanese children under 7 years old and their mothers to pain caused by inoculation of the children. Under the age of 5 crying was a typical child behaviour, whereas 5- and 6-year-olds showed facial expressions instead. Their mothers were generally empathetic to the children’s distress on inoculation, but the empathetic reactions tended to decrease as the children grew older. However, children’s crying per se did not necessarily cause the reactions, and the mothers of the older children tended to react to the children’s pain with a smile. A greater proportion of 3-year-old females cried than males of the same age, but despite this their mothers had a stronger tendency to respond with a smile in comparison with the mothers of male children. The results were discussed with respect to development of emotional independence and competence of social manipulation by children.
Developmental Maturity Expectations of Israeli Fathers and Mothers: Effects of Education, Ethnic Origin, and Religiosity
Authors: Mansbach Ivonne Kleinfeld , Greenbaum Charles W.
Published: July 2002

This study investigated expectations of maturity in children among 380 Israeli Jewish fathers and mothers of 6-month-old infants. Subjects were administered a questionnaire which included items reflecting three developmental domains: Cognitive Maturity, Biosocial Maturity, and Social Autonomy. Regarding Cognitive Maturity mothers had earlier expectations than fathers, intermediate and higher educated parents had earlier expectations than lower educated and nonreligious (Secular) parents and moderately religious (Traditional) parents had earlier maturity expectations than religiously strict (Orthodox) parents. A reverse pattern could be observed for Biosocial Maturity: lower educated parents had earlier expectations than higher educated parents, Orthodox parents had earlier expectations than Traditional or Secular parents and parents of Asian or North African origin had earlier maturity expectations than parents of European or American origin. On Social Autonomy mothers had earlier maturity expectations than fathers and Secular and Orthodox parents had earlier expectations than Traditional parents. The results suggest that parental expectations of maturity form a multidimensional construct in which sex and social background of the parent are related to early expectations in some areas and late expectations in others, depending on the values of their societal subgroup.
Elasticity and Structure of Eukaryote Chromosomes Studied by Micromanipulation and Micropipette Aspiration
Authors: Houchmandzadeh Bahram , Marko John F. , Chatenay Didier , Libchaber Albert
Published: July 2002

The structure of mitotic chromosomes in cultured newt lung cells was investigated by a quantitative study of their deformability, using micropipettes. Metaphase chromosomes are highly extensible objects that return to their native shape after being stretched up to 10 times their normal length. Larger deformations of 10 to 100 times irreversibly and progressively transform the chromosomes into a “thin filament,” parts of which display a helical organization. Chromosomes break for elongations of the order of 100 times, at which time the applied force is around 100 nanonewtons. We have also observed that as mitosis proceeds from nuclear envelope breakdown to metaphase, the native chromosomes progressively become more flexible. (The elastic Young modulus drops from 5,000 ± 1,000 to 1,000 ± 200 Pa.) These observations and measurements are in agreement with a helix-hierarchy model of chromosome structure. Knowing the Young modulus allows us to estimate that the force exerted by the spindle on a newt chromosome at anaphase is roughly one nanonewton.
Neurotrophin-3–enhanced Nerve Regeneration Selectively Improves Recovery of Muscle Fibers Expressing Myosin Heavy Chains 2b
Authors: Sterne G.D. , Coulton G.R. , Brown R.A. , Green C.J. , Terenghi G.
Published: July 2002

The purpose of this study was to evaluate the effect of neurotrophin 3 (NT-3) enhanced nerve regeneration on the reinnervation of a target muscle. Muscle fibers can be classified according to their mechanical properties and myosin heavy chain (MHC) isoform composition. MHC1 containing slow-type and MHC2a or 2b fast-type fibers are normally distributed in a mosaic pattern, their phenotype dictated by motor innervation. After denervation, all fibers switch to fast-type MHC2b expression and also undergo atrophy resulting in loss of muscle mass. After regeneration, discrimination between fast and slow fibers returns, but the distribution and fiber size change according to the level of reinnervation.

In this study, rat gastrocnemius muscles (ipsilateral and contralateral to the side of nerve injury) were collected up to 8 mo after nerve repair, with or without local delivery of NT-3. The phenotype changes of MHC1, 2a, and 2b were analyzed by immunohistochemistry, and fiber type proportion, diameter, and grouping were assessed by computerized image analysis. At 8 mo, the local delivery of NT-3 resulted in significant improvement in gastrocnemius muscle weight compared with controls (NT-3 group 47%, controls 39% weight of contralateral normal muscle; P < 0.05). NT-3 delivery resulted in a significant increase in the proportion (NT-3 43.3%, controls 35.7%; P < 0.05) and diameter (NT-3 87.8 μm, controls 70.8 μm; P < 0.05) of fast type 2b fibers after reinnervation. This effect was specific to type 2b fibers; no normalization was seen in other fiber types.

This study indicates that NT-3–enhanced axonal regeneration has a beneficial effect on the motor target organ. Also, NT-3 may be specifically affecting a subset of motoneurons that determine type 2b muscle fiber phenotype. As NT-3 was topically applied to cut nerves, our data suggest a discriminating effect of the neurotrophin on neuro–muscular interaction. These results would imply that muscle fibers may be differentially responsive to other neurotrophic factors and indicate the potential clinical role of NT-3 in the prevention of muscle atrophy after nerve injury.
Roles of LAP2 Proteins in Nuclear Assembly and DNA Replication: Truncated LAP2β Proteins Alter Lamina Assembly, Envelope Formation, Nuclear Size, and DNA Replication Efficiency in Xenopus laevis Extracts
Authors: Gant Tracey Michele , Harris Crafford A. , Wilson Katherine L.
Published: July 2002

Humans express three major splicing isoforms of LAP2, a lamin- and chromatin-binding nuclear protein. LAP2β and γ are integral membrane proteins, whereas α is intranuclear. When truncated recombinant human LAP2β proteins were added to cell-free Xenopus laevis nuclear assembly reactions at high concentrations, a domain common to all LAP2 isoforms (residues 1–187) inhibited membrane binding to chromatin, whereas the chromatin- and lamin-binding region (residues 1–408) inhibited chromatin expansion. At lower concentrations of the common domain, membranes attached to chromatin with a unique scalloped morphology, but these nuclei neither accumulated lamins nor replicated. At lower concentrations of the chromatin- and lamin-binding region, nuclear envelopes and lamins assembled, but nuclei failed to enlarge and replicated on average 2.5-fold better than controls. This enhancement was not due to rereplication, as shown by density substitution experiments, suggesting the hypothesis that LAP2β is a downstream effector of lamina assembly in promoting replication competence. Overall, our findings suggest that LAP2 proteins mediate membrane–chromatin attachment and lamina assembly, and may promote replication by influencing chromatin structure.
Myosin Light Chain Kinase Functions Downstream of Ras/ERK to Promote Migration of Urokinase-Type Plasminogen Activator-Stimulated Cells in an Integrin-Selective Manner
Authors: Nguyen Diem H.D. , Catling Andrew D. , Webb Donna J. , Sankovic Mauricio , Walker Lori A. , Somlyo Avril V. , Weber Michael J. , Gonias Steven L.
Published: July 2002

Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a β1-integrin (probably αVβ1) and αVβ5. When MCF-7 cells were transfected to express αVβ3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of αVβ3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.
α-Bungarotoxin Receptors Contain α7 Subunits in Two Different Disulfide-Bonded Conformations
Authors: Rakhilin Sergey , Drisdel Renaldo C. , Sagher Daphna , McGehee Daniel S. , Vallejo Yolanda , Green William N.
Published: July 2002

Neuronal nicotinic α7 subunits assemble into cell-surface complexes that neither function nor bind α-bungarotoxin when expressed in tsA201 cells. Functional α-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the α7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric α7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, α7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second α7/serotonin-3 subunit conformation coincides with the formation of α-bungarotoxin–binding sites and intrasubunit disulfide bonding, apparently within the α7 domain of the α7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional α7 receptors, α7 subunits undergo a conformational change similar to α7/serotonin-3 subunits. α7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, α-bungarotoxin–binding receptors with two α7 conformations. Our results suggest that α7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.
Selective Uncoupling of P120ctn from E-Cadherin Disrupts Strong Adhesion
Authors: Thoreson Molly A. , Anastasiadis Panos Z. , Daniel Juliet M. , Ireton Reneé C. , Wheelock Margaret J. , Johnson Keith R. , Hummingbird Diana K. , Reynolds Albert B.
Published: July 2002

p120ctn is a catenin whose direct binding to the juxtamembrane domain of classical cadherins suggests a role in regulating cell–cell adhesion. The juxtamembrane domain has been implicated in a variety of roles including cadherin clustering, cell motility, and neuronal outgrowth, raising the possibility that p120 mediates these activities. We have generated minimal mutations in this region that uncouple the E-cadherin–p120 interaction, but do not affect interactions with other catenins. By stable transfection into E-cadherin–deficient cell lines, we show that cadherins are both necessary and sufficient for recruitment of p120 to junctions. Detergent-free subcellular fractionation studies indicated that, in contrast to previous reports, the stoichiometry of the interaction is extremely high. Unlike α- and β-catenins, p120 was metabolically stable in cadherin-deficient cells, and was present at high levels in the cytoplasm. Analysis of cells expressing E-cadherin mutant constructs indicated that p120 is required for the E-cadherin–mediated transition from weak to strong adhesion. In aggregation assays, cells expressing p120-uncoupled E-cadherin formed only weak cell aggregates, which immediately dispersed into single cells upon pipetting. As an apparent consequence, the actin cytoskeleton failed to insert properly into peripheral E-cadherin plaques, resulting in the inability to form a continuous circumferential ring around cell colonies. Our data suggest that p120 directly or indirectly regulates the E-cadherin–mediated transition to tight cell–cell adhesion, possibly blocking subsequent events necessary for reorganization of the actin cytoskeleton and compaction.
TRP and the PDZ Protein, Inad, Form the Core Complex Required for Retention of the Signalplex in Drosophila Photoreceptor Cells
Authors: Li Hong-Sheng , Montell Craig
Published: July 2002

The light response in Drosophila photoreceptor cells is mediated by a series of proteins that assemble into a macromolecular complex referred to as the signalplex. The central player in the signalplex is inactivation no afterpotential D (INAD), a protein consisting of a tandem array of five PDZ domains. At least seven proteins bind INAD, including the transient receptor potential (TRP) channel, which depends on INAD for localization to the phototransducing organelle, the rhabdomere. However, the determinants required for localization of INAD are not known. In this work, we showed that INAD was required for retention rather than targeting of TRP to the rhabdomeres. In addition, we demonstrated that TRP bound to INAD through the COOH terminus, and this interaction was required for localization of INAD. Other proteins that depend on INAD for localization, phospholipase C and protein kinase C, also mislocalized. However, elimination of any other member of the signalplex had no impact on the spatial distribution of INAD. A direct interaction between TRP and INAD did not appear to have a role in the photoresponse independent of localization of multiple signaling components. Rather, the primary function of the TRP/ INAD complex is to form the core unit required for localization of the signalplex to the rhabdomeres.
The N domain of Smad7 is essential for specific inhibition of transforming growth factor-β signaling
Authors: Hanyu Aki , Ishidou Yasuhiro , Ebisawa Takanori , Shimanuki Tomomasa , Imamura Takeshi , Miyazono Kohei
Published: July 2002

Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-β (TGF-β) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-β and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-β signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-β and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-β signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-β type I receptor (TβR-I) more efficiently, and were more potent in repressing TGF-β signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-β receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-β signaling.
Interleukin 12 (IL-12) Is Crucial to the Development of Protective Immunity in Mice Intravenously Infected with Mycobacterium tuberculosis
Authors: Cooper Andrea M. , Magram Jeanne , Ferrante Jessica , Orme Ian M.
Published: July 2002

Immunity to Mycobacterium tuberculosis infection is associated with the emergence of protective CD4 T cells that secrete cytokines, resulting in activation of macrophages and the recruitment of monocytes to initiate granuloma formation. The cytokine-mediating macrophage activation is interferon-γ (IFN-γ), which is largely dependent on interleukin-12 (IL-12) for its induction. To address the role of IL-12 in immunity to tuberculosis, IL-12 p40−/− mice were infected with M. tuberculosis and their capacity to control bacterial growth and other characteristics of their immune response were determined. The IL-12 p40−/− mice were unable to control bacterial growth and this appeared to be linked to the absence of both innate and acquired sources of IFN-γ. T cell activation as measured by delayed type hypersensitivity and lymphocyte accumulation at the site of infection were both markedly reduced in the IL-12 p40−/− mice. Therefore, IL-12 is essential to the generation of a protective immune response to M. tuberculosis, with its main functions being the induction of the expression of IFN-γ and the activation of antigen-specific lymphocytes capable of creating a protective granuloma.