bluuebunny/crossref_metadata_2025 huggingface dataset:
Survival motor neuron protein modulates neuron-specific apoptosis
Authors: Kerr Douglas A. , Nery Jocelyn P. , Traystman Richard J. , Chau B. Nelson , Hardwick J. Marie
Published: July 2002

Spinal muscular atrophy (SMA) is attributed to mutations in the
SMN1
gene, leading to loss of spinal cord motor neurons.
The neurotropic Sindbis virus vector system was used to investigate a
role for the survival motor neuron (SMN) protein in regulating neuronal
apoptosis. Here we show that SMN protects primary neurons and
differentiated neuron-like stem cells, but not cultured cell lines from
virus-induced apoptotic death. SMN also protects neurons
in vivo
and increases survival of virus-infected mice.
SMN mutants (SMNΔ7 and SMN-Y272C) found in patients with SMA not only
lack antiapoptotic activity but also are potently
proapoptotic, causing increased neuronal apoptosis and
animal mortality. Full-length SMN is proteolytically processed in
brains undergoing apoptosis or after ischemic injury. Mutation
of an Asp-252 of SMN abolished cleavage of SMN and increased the
antiapoptotic function of full-length SMN in neurons. Taken
together, deletions or mutations of the C terminus of SMN that result
from proteolysis, splicing (SMNΔ7), or germ-line mutations (e.g.,
Y272C), produce a proapoptotic form of SMN that may contribute
to neuronal death in SMA and perhaps other neurodegenerative disorders.
Disruption of the neural response to rapid acoustic stimuli in dyslexia: Evidence from functional MRI
Authors: Temple E. , Poldrack R. A. , Protopapas A. , Nagarajan S. , Salz T. , Tallal P. , Merzenich M. M. , Gabrieli J. D. E.
Published: July 2002

The biological basis for developmental dyslexia remains unknown.
Research has suggested that a fundamental deficit in dyslexia is the
inability to process sensory input that enters the nervous system
rapidly and that deficits in processing rapid acoustic information are
associated with impaired reading. Functional magnetic resonance imaging
(fMRI) was used to identify the brain basis of rapid acoustic
processing in normal readers and to discover the status of that
response in dyslexic readers. Normal readers showed left prefrontal
activity in response to rapidly changing, relative to slowly changing,
nonlinguistic acoustic stimuli. Dyslexic readers showed no differential
left frontal response. Two dyslexic readers participated in a
remediation program and showed increased activity in left prefrontal
cortex after training. These fMRI results identify left prefrontal
regions as normally being sensitive to rapid relative to slow acoustic
stimulation, insensitive to the difference between such stimuli in
dyslexic readers, and plastic enough in adulthood to develop such
differential sensitivity after intensive training.
Variable deletion and duplication at recombination junction ends: Implication for staggered double-strand cleavage in class-switch recombination
Authors: Chen Xiaochuan , Kinoshita Kazuo , Honjo Tasuku
Published: July 2002

Immunoglobulin class-switch recombination (CSR) gives rise to looped-out circular DNA of a cleaved S segment, which is lost eventually after cell divisions. To understand the molecular mechanism of S region cleavage during CSR, we constructed artificial CSR substrates in which inversion-type CSR takes place to retain the cleaved S segment. Sequencing analyses of recombinant clones of these substrates revealed that varying degrees of deletions and duplications exist at CSR breakpoints, suggesting the involvement of staggered cleavage of the S region in CSR. In addition, mutations frequently found near junctions showed a similar profile of base replacement to Ig somatic hypermutation. These findings suggest that single-strand tails of staggered cleavage may be repaired by error-prone DNA synthesis.
Evidence that two present-day components needed for the genetic code appeared after nucleated cells separated from eubacteria.
Authors: Ribas de Pouplana L , Frugier M , Quinn C L , Schimmel P
Published: July 2002

The trinucleotide/amino acid relationships of the present-day genetic code are established by the amino-acylation reactions of tRNA synthetases, whereby each of 20 specific amino acids is attached to its cognate tRNAs, which bear anticodon trinucleotides. Because of its universality, the appearance of the modern genetic code is thought to predate the separation of prokaryotic and eukaryotic organisms in the universal phylogenetic tree. In the light of new sequence information, we present here a phylogenetic analysis that shows an unusual picture for tyrosyl- and tryptophanyl-tRNA synthetases. Ij particular, the eukaryotic tyrosyl- and tryptophanyl-tRNA synthetases are more related to each other than to their respective prokaryotic counterparts. In contrast, each of the other 18 eukaryotic synthetases is more related to its prokaryotic counterpart than to any eukaryotic synthetase specific for a different amino acid. Our results raise the possibility that present day tyrosyl- and tryptophanyl-tRNA synthetases appeared after the separation of nucleated cells from eubacteria. The results have implications for the development of the genetic code.
A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells.
Authors: Su H , Raymond L , Rockey D D , Fischer E , Hackstadt T , Caldwell H D
Published: July 2002

Chlamydial attachment to columnar conjunctival or urogenital epithelial cells is an initial and critical step in the pathogenesis of chlamydial mucosal infections. The chlamydial major outer membrane protein (MOMP) has been implicated as a putative chlamydial cytoadhesin; however, direct evidence supporting this hypothesis has not been reported. The function of MOMP as a cytoadhesin was directly investigated by expressing the protein as a fusion with the Escherichia coli maltose binding protein (MBP-MOMP) and studying its interaction with human epithelial cells. The recombinant MBP-MOMP bound specifically to HeLa cells at 4 degrees C but was not internalized after shifting the temperature to 37 degrees C. The MBP-MOMP competitively inhibited the infectivity of viable chlamydiae for epithelial cells, indicating that the MOMP and intact chlamydiae bind the same host receptor. Heparan sulfate markedly reduced binding of the MBP-MOMP to cells, whereas chondroitin sulfate had no effect on binding. Enzymatic treatment of cells with heparitinase but not chondroitinase inhibited the binding of MBP-MOMP. These same treatments were also shown to reduce the infectivity of chlamydiae for epithelial cells. Mutant cell lines defective in heparan sulfate synthesis but not chondroitin sulfate synthesis showed a marked reduction in the binding of MBP-MOMP and were also less susceptible to infection by chlamydiae. Collectively, these findings provide strong evidence that the MOMP functions as a chlamydial cytoadhesin and that heparan sulfate proteoglycans are the host-cell receptors to which the MOMP binds.
Muscarinic acetylcholine receptor expression in memory circuits: Implications for treatment of Alzheimer disease
Authors: Levey Allan I.
Published: July 2002

Cholinergic transmission at muscarinic acetylcholine receptors
(mAChR) has been implicated in higher brain functions such as learning
and memory, and loss of synapses may contribute to the symptoms of
Alzheimer disease. A heterogeneous family of five genetically distinct
mAChR subtypes differentially modulate a variety of intracellular
signaling systems as well as the processing of key molecules involved
in the pathology of the disease. Although many muscarinic effects have
been identified in memory circuits, including a diversity of pre- and
post-synaptic actions in hippocampus, the identities of the molecular
subtypes responsible for any given function remain elusive. All five
mAChR genes are expressed in hippocampus, and subtype-specific
antibodies have enabled identification, quantification, and
localization of the encoded proteins. The m1, m2, and m4 mAChR proteins
are most abundant in forebrain regions and they have distinct cellular
and subcellular localizations suggestive of various pre- and
postsynaptic functions in cholinergic circuits. The subtypes are also
differentially altered in postmortem brain samples from Alzheimer
disease cases. Further understanding of the molecular pharmacology of
failing synapses in Alzheimer disease, together with the development of
new subtype-selective drugs, may provide more specific and effective
treatments for the disease.
Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90
Authors: Schneider Christine , Sepp-Lorenzino Laura , Nimmesgern Elmar , Ouerfelli Ouathek , Danishefsky Samuel , Rosen Neal , Hartl F. Ulrich
Published: July 2002

The role of the abundant stress protein Hsp90 in protecting cells
against stress-induced damage is not well understood. The recent
discovery that a class of ansamycin antibiotics bind specifically to
Hsp90 allowed us to address this problem from a new angle. We find that
mammalian Hsp90, in cooperation with Hsp70, p60, and other factors,
mediates the ATP-dependent refolding of heat-denatured proteins, such
as firefly luciferase. Failure to refold results in proteolysis. The
ansamycins inhibit refolding, both
in vivo
and in a cell
extract, by preventing normal dissociation of Hsp90 from luciferase,
causing its enhanced degradation. This mechanism also explains the
ansamycin-induced proteolysis of several protooncogenic protein
kinases, such as Raf-1, which interact with Hsp90. We propose that
Hsp90 is part of a quality control system that facilitates protein
refolding or degradation during recovery from stress. This function is
used by a limited set of signal transduction molecules for their
folding and regulation under nonstress conditions. The ansamycins shift
the mode of Hsp90 from refolding to degradation, and this effect is
probably amplified for specific Hsp90 substrates.
Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis
Authors: Landino Lisa M. , Crews Brenda C. , Timmons Michael D. , Morrow Jason D. , Marnett Lawrence J.
Published: July 2002

Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes’ peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or byin situgeneration of peroxynitrite from NO coupling to superoxide anion. Cu,Zn-superoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generatedin situfrom superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid)2, and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inflammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.
Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid beta at the blood-brain and blood-cerebrospinal fluid barriers.
Authors: Zlokovic B V , Martel C L , Matsubara E , McComb J G , Zheng G , McCluskey R T , Frangione B , Ghiso J
Published: July 2002

A soluble form of Alzheimer disease amyloid beta-protein (sA beta) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA beta (1-40)-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA beta (1-40)-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA beta (1-40)-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA beta (1-40)-apoJ complex at the cerebral vascular endothelium and choroid epithelium.
Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor
Authors: Holgado-Madruga Marina , Moscatello David K. , Emlet David R. , Dieterich Rebekka , Wong Albert J.
Published: July 2002

Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF.
Hydrophobic sequence minimization of the α-lactalbumin molten globule
Authors: Wu Lawren C. , Kim Peter S.
Published: July 2002

The molten globule, a widespread protein-folding intermediate, can attain a native-like backbone topology, even in the apparent absence of rigid side-chain packing. Nonetheless, mutagenesis studies suggest that molten globules are stabilized by some degree of side-chain packing among specific hydrophobic residues. Here we investigate the importance of hydrophobic side-chain diversity in determining the overall fold of the α-lactalbumin molten globule. We have replaced all of the hydrophobic amino acids in the sequence of the helical domain with a representative amino acid, leucine. Remarkably, the minimized molecule forms a molten globule that retains many structural features characteristic of a native α-lactalbumin fold. Thus, nonspecific hydrophobic interactions may be sufficient to determine the global fold of a protein.
RanBP2 associates with Ubc9p and a modified form of RanGAP1
Authors: Saitoh Hisato , Pu Robert , Cavenagh Margaret , Dasso Mary
Published: July 2002

Ran is a small GTPase required for nuclear transport in eukaryotic cells [Gorlich, D. & Mattaj, I. W. (1996)
Science
271, 1513–1518]. Mutants in Ran also show defects in mRNA processing, cell cycle regulation, and other aspects of nuclear function [Rush, M. G., Drivas, G. & D’Eustachio, P. (1996)
BioEssays
18, 103–112; Sazer, S. (1996)
Trends Cell Biol.
6, 81–85]. In an effort to understand the role of Ran in these diverse processes, we previously characterized 10 Ran interacting proteins (Rips) from
Xenopus
egg extracts. In this report, we present further characterization of a complex containing three of these Rips: p340
RanBP2
, p88, and p18. We have cloned the
Xenopus
homologue of RanGAP1, and we show here that p88 is a modified form of this protein. In RanGAP assays, the p340
RanBP2
–p88–p18 complex contains GTPase-activating protein activity, indicating that RanGAP1 is not inactivated by modification. Rather, modification of RanGAP1 appears to be linked to its association with p340
RanBP2
because we did not observe unmodified RanGAP1 in p340
RanBP2
immunoprecipitates. We have also characterized p18, and we found that it is the
Xenopus
homologue of Ubc9p, an E2 ubiquitin-conjugating enzyme that is required for cell cycle regulation [Seufert, W., Futcher, B. & Jentsch, S. (1995)
Nature (London)
373, 78–81]. Using antibodies directed against
Xenopus
Ubc9p, we have confirmed that Ubc9p associates with p340
RanBP2
in
Xenopus
extracts. These results suggest Ubc9p’s role in cell cycle regulation may involve either modification of nuclear transport substrates or the nuclear transport machinery.
Amyloid β-peptide stimulates nitric oxide production in astrocytes through an NFκB-dependent mechanism
Authors: Akama Keith T. , Albanese Chris , Pestell Richard G. , Van Eldik Linda J.
Published: July 2002

The major pathological features of Alzheimer’s disease (AD) include amyloid plaques composed primarily of the β-amyloid (Aβ) peptide, degenerating neurons and neurofibrillary tangles, and the presence of numerous activated astrocytes and microglia. Although extensive genetic data implicate Aβ in the neurodegenerative cascade of AD, the molecular mechanisms underlying its effects on neurons and glia and the relationship between glial activation and neuronal death are not well defined. Aβ has been shown to induce glial activation, and a growing body of evidence suggests that activated glia contribute to neurotoxicity through generation of inflammatory cytokines and neurotoxic free radicals, such as nitric oxide (NO), potent sources of oxidative stress known to occur in AD. It is therefore crucial to identify specific Aβ-induced molecular pathways mediating these responses in activated glia. We report that Aβ stimulates the activation of the transcription factor NFκB in rat astrocytes, that NFκB activation occurs selectively from p65 transactivation domain 2, and that Aβ-induced NO synthase expression and NO production occur through an NFκB-dependent mechanism. This demonstration of how Aβ couples an intracellular signal transduction pathway involving NFκB to a potentially neurotoxic response provides a key mechanistic link between Aβ and the generation of oxidative damage. Our results also suggest possible molecular targets upon which to focus future drug discovery efforts for AD.
Mutations in the chloride-bicarbonate exchanger gene
AE1
cause autosomal dominant but not autosomal recessive distal renal tubular acidosis
Authors: Karet F. E. , Gainza F. J. , Györy A. Z. , Unwin R. J. , Wrong O. , Tanner M. J. A. , Nayir A. , Alpay H. , Santos F. , Hulton S. A. , Bakkaloglu A. , Ozen S. , Cunningham M. J. , di Pietro A. , Walker W. G. , Lifton R. P.
Published: July 2002

Primary distal renal tubular acidosis (dRTA) is characterized by reduced ability to acidify urine, variable hyperchloremic hypokalemic metabolic acidosis, nephrocalcinosis, and nephrolithiasis. Kindreds showing either autosomal dominant or recessive transmission are described. Mutations in the chloride-bicarbonate exchanger
AE1
have recently been reported in four autosomal dominant dRTA kindreds, three of these altering codon Arg589. We have screened 26 kindreds with primary dRTA for mutations in
AE1
. Inheritance was autosomal recessive in seventeen kindreds, autosomal dominant in one, and uncertain due to unknown parental phenotype or sporadic disease in eight kindreds. No mutations in
AE1
were detected in any of the autosomal recessive kindreds, and analysis of linkage showed no evidence of linkage of recessive dRTA to
AE1
. In contrast, heterozygous mutations in
AE1
were identified in the one known dominant dRTA kindred, in one sporadic case, and one kindred with two affected brothers. In the dominant kindred, the mutation Arg-589/Ser cosegregated with dRTA in the extended pedigree. An Arg-589/His mutation in the sporadic case proved to be a
de novo
mutation. In the third kindred, affected brothers both have an intragenic 13-bp duplication resulting in deletion of the last 11 amino acids of AE1. These mutations were not detected in 80 alleles from unrelated normal individuals. These findings underscore the key role of Arg-589 and the C terminus in normal AE1 function, and indicate that while mutations in
AE1
cause autosomal dominant dRTA, defects in this gene are not responsible for recessive disease.
Crystal structure of 2,5-diketo-
d
-gluconic acid reductase A complexed with NADPH at 2.1-Å resolution
Authors: Khurana Sumit , Powers David B. , Anderson Stephen , Blaber Michael
Published: July 2002

The three-dimensional structure of
Corynebacterium
2,5-diketo-
d
-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-Å resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-
d
-gluconate (2,5-DKG) to 2-keto-
l
-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo–keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of
l
-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-
l
-gulonate from
d
-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.
Mutation in the tau gene in familial multiple system tauopathy with presenile dementia
Authors: Spillantini Maria Grazia , Murrell Jill R. , Goedert Michel , Farlow Martin R. , Klug Aaron , Ghetti Bernardino
Published: July 2002

Familial multiple system tauopathy with presenile dementia (MSTD) is a neurodegenerative disease with an abundant filamentous tau protein pathology. It belongs to the group of familial frontotemporal dementias with Parkinsonism linked to chromosome 17 (FTDP-17), a major class of inherited dementing disorders whose genetic basis is unknown. We now report a G to A transition in the intron following exon 10 of the gene for microtubule-associated protein tau in familial MSTD. The mutation is located at the 3′ neighboring nucleotide of the GT splice-donor site and disrupts a predicted stem-loop structure. We also report an abnormal preponderance of soluble tau protein isoforms with four microtubule-binding repeats over isoforms with three repeats in familial MSTD. This most likely accounts for our previous finding that sarkosyl-insoluble tau protein extracted from the filamentous deposits in familial MSTD consists only of tau isoforms with four repeats. These findings reveal that a departure from the normal ratio of four-repeat to three-repeat tau isoforms leads to the formation of abnormal tau filaments. The results show that dysregulation of tau protein production can cause neurodegeneration and imply that the FTDP-17 gene is the tau gene. This work has major implications for Alzheimer’s disease and other tauopathies.
Protein thermostability above 100°C: A key role for ionic interactions
Authors: Vetriani Costantino , Maeder Dennis L. , Tolliday Nicola , Yip Kitty S.-P. , Stillman Timothy J. , Britton K. Linda , Rice David W. , Klump Horst H. , Robb Frank T.
Published: July 2002

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100°C. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles
Pyrococcus furiosus
and
Thermococcus litoralis
whose optimal growth temperatures are 100°C and 88°C, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104°C. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from
T. litoralis
, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104°C over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.
Derivation of pluripotent stem cells from cultured human primordial germ cells
Authors: Shamblott Michael J. , Axelman Joyce , Wang Shunping , Bugg Elizabeth M. , Littlefield John W. , Donovan Peter J. , Blumenthal Paul D. , Huggins George R. , Gearhart John D.
Published: July 2002

Human pluripotent stem cells would be invaluable forin vitrostudies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5–9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7–21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.
Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy
Authors: Conway J. F. , Cheng N. , Zlotnick A. , Stahl S. J. , Wingfield P. T. , Steven A. C.
Published: July 2002

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 Å, enabling direct visualization of α-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i.e., insertion of peptides, ≈10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 Å resolution. Hepatitis B virus capsids are icosahedral particles, ≈300 Å in diameter, made up of T-shaped dimers (subunit
M
r
,
16–21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and −7 (in the residual propeptide) in the “e-antigen” form of the capsid protein and has implications for why this clinically important antigen remains unassembled
in vivo
.
Frequent occurrence of deletions and duplications during somatic hypermutation: Implications for oncogene translocations and heavy chain disease
Authors: Goossens Tina , Klein Ulf , Küppers Ralf
Published: July 2002

Human naive and germinal center (GC) B cells were sorted by flow cytometry and rearranged VHregion genes were amplified and sequenced from single cells. Whereas no deletions or insertions were found in naive B cells, ≈4% of in-frame and >40% of out-of-frame rearrangements of GC B cells harbored deletions and/or insertions of variable length. The pattern of deletions/insertions and their restriction to mutated V genes strongly suggests that they result from somatic hypermutation. Deletions and insertions account for ≈6% of somatic mutations introduced into rearranged VHregion genes of GC B cells. These deletions/insertions seem to be the main cause for the generation of heavy chain disease proteins. Furthermore, it appears that several types of oncogene translocations (like c-myc translocations in Burkitt’s lymphoma) occur as a byproduct of somatic hypermutation within the GC—and not during V(D)J recombination in the bone marrow as previously thought.
A role for Pak protein kinases in Schwann cell transformation
Authors: Tang Yi , Marwaha Sunil , Rutkowski J. Lynn , Tennekoon Gihan I. , Phillips Peter C. , Field Jeffrey
Published: July 2002

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by loss of the NF1 gene, is characterized clinically by neurofibromas and more rarely by neurofibrosarcomas. Neurofibromin, the protein encoded by NF1, possesses an intrinsic GTPase accelerating activity for the Ras proto-oncogene. Through this activity, it is a negative regulator of Ras. The Pak protein kinase is a candidate for a downstream signaling protein that may mediate Ras signals because it is activated by Rac and Cdc42, two small G proteins required for Ras signaling. Here, we use Pak mutants to explore the role of Pak in Ras signaling in Schwann cells, the cells affected in NF1. Whereas an activated Pak mutant does not transform cells, dominant negative Pak mutants are potent inhibitors of Ras transformation of rat Schwann cells and of a neurofibrosarcoma cell line from an NF1 patient. Although activated Pak stimulated jun-N-terminal kinase, inhibition of Ras transformation by dominant negative Pak did not require inhibition of jun-N-terminal kinase. Instead, the Pak mutants appeared to inhibit transformation by preventing Ras activation of the ERK/mitogen-activated protein kinase cascade. These results have implications for our understanding of NF1 because a neurofibrosarcoma cell line derived from a patient with NF1 was reverted by stable expression of the Pak dominant negative mutants.
Molecular analysis of
NOZZLE
, a gene involved in pattern formation and early sporogenesis during sex organ development in
Arabidopsis thaliana
Authors: Schiefthaler Ursula , Balasubramanian Sureshkumar , Sieber Patrick , Chevalier David , Wisman Ellen , Schneitz Kay
Published: July 2002

Sexual reproduction is a salient aspect of plants, and elaborate structures, such as the flowers of angiosperms, have evolved that aid in this process. Within the flower the corresponding sex organs, the anther and the ovule, form the male and female sporangia, the pollen sac and the nucellus, respectively. However, despite their central role for sexual reproduction little is known about the mechanisms that control the establishment of these important structures. Here we present the identification and molecular characterization of the
NOZZLE
(
NZZ
) gene in the flowering plant
Arabidopsis thaliana
. In several
nzz
mutants the nucellus and the pollen sac fail to form. It indicates that
NZZ
plays an early and central role in the development of both types of sporangia and that the mechanisms controlling these processes share a crucial factor. In addition,
NZZ
may have an early function during male and female sporogenesis as well. The evolutionary aspects of these findings are discussed.
NZZ
encodes a putative protein of unknown function. However, based on sequence analysis we speculate that NZZ is a nuclear protein and possibly a transcription factor.
Segregating the functions of human hippocampus
Authors: Strange B. A. , Fletcher P. C. , Henson R. N. A. , Friston K. J. , Dolan R. J.
Published: July 2002

It is now accepted that hippocampal lesions impair episodic memory. However, the precise functional role of the hippocampus in episodic memory remains elusive. Recent functional imaging data implicate the hippocampus in processing novelty, a finding supported by human
in vivo
recordings and event-related potential studies. Here we measure hippocampal responses to novelty, using functional MRI (fMRI), during an item-learning paradigm generated from an artificial grammar system. During learning, two distinct types of novelty were periodically introduced: perceptual novelty, pertaining to the physical characteristics of stimuli (in this case visual characteristics), and exemplar novelty, reflecting semantic characteristics of stimuli (in this case grammatical status within a rule system). We demonstrate a left anterior hippocampal response to both types of novelty and adaptation of these responses with stimulus familiarity. By contrast to these novelty effects, we also show bilateral posterior hippocampal responses with increasing exemplar familiarity. These results suggest a functional dissociation within the hippocampus with respect to the relative familiarity of study items. Neural responses in anterior hippocampus index generic novelty, whereas posterior hippocampal responses index familiarity to stimuli that have behavioral relevance (i.e., only exemplar familiarity). These findings add to recent evidence for functional segregation within the human hippocampus during learning.
Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds
Authors: Holmes Todd C. , de Lacalle Sonsoles , Su Xing , Liu Guosong , Rich Alexander , Zhang Shuguang
Published: July 2002

A new type of
s
elf-
a
ssembling
peptide
(sapeptide) scaffolds that serve as substrates
for neurite outgrowth and synapse formation is described. These
peptide-based scaffolds are amenable to molecular design by using
chemical or biotechnological syntheses. They can be tailored to a
variety of applications. The sapeptide scaffolds are formed through the
spontaneous assembly of ionic self-complementary β-sheet
oligopeptides under physiological conditions, producing a hydrogel
material. The scaffolds can support neuronal cell attachment and
differentiation as well as extensive neurite outgrowth. Furthermore,
they are permissive substrates for functional synapse formation between
the attached neurons. That primary rat neurons form active synapses on
such scaffold surfaces
in situ
suggests these scaffolds
could be useful for tissue engineering applications. The buoyant
sapeptide scaffolds with attached cells in culture can be transported
readily from one environment to another. Furthermore, these peptides
did not elicit a measurable immune response or tissue inflammation when
introduced into animals. These biological materials created through
molecular design and self assembly may be developed as a biologically
compatible scaffold for tissue repair and tissue engineering.
An approach to probe some neural systems interaction by functional MRI at neural time scale down to milliseconds
Authors: Ogawa Seiji , Lee Tso-Ming , Stepnoski Ray , Chen Wei , Zhu Xiao-Hong , Ugurbil Kamil
Published: July 2002

In this paper, we demonstrate an approach by which some evoked
neuronal events can be probed by functional MRI (fMRI) signal with
temporal resolution at the time scale of tens of milliseconds. The
approach is based on the close relationship between neuronal electrical
events and fMRI signal that is experimentally demonstrated in
concurrent fMRI and electroencephalographic (EEG) studies conducted in
a rat model with forepaw electrical stimulation. We observed a
refractory period of neuronal origin in a two-stimuli paradigm: the
first stimulation pulse suppressed the evoked activity in both EEG and
fMRI signal responding to the subsequent stimulus for a period of
several hundred milliseconds. When there was an apparent site–site
interaction detected in the evoked EEG signal induced by two stimuli
that were primarily targeted to activate two different sites in the
brain, fMRI also displayed signal amplitude modulation because of the
interactive event. With visual stimulation using two short pulses in
the human brain, a similar refractory phenomenon was observed in
activated fMRI signals in the primary visual cortex. In addition, for
interstimulus intervals shorter than the known latency time of the
evoked potential induced by the first stimulus (≈100 ms) in the
primary visual cortex of the human brain, the suppression was not
present. Thus, by controlling the temporal relation of input tasks, it
is possible to study temporal evolution of certain neural events at the
time scale of their evoked electrical activity by noninvasive fMRI
methodology.
The mechanism of substrate (aglycone) specificity in β-glucosidases is revealed by crystal structures of mutant maize β-glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes
Authors: Czjzek Mirjam , Cicek Muzaffer , Zamboni Véronique , Bevan David R. , Henrissat Bernard , Esen Asim
Published: July 2002

The mechanism and the site of substrate (i.e., aglycone)
recognition and specificity were investigated in maize β-glucosidase
(Glu1) by x-ray crystallography by using crystals of a catalytically
inactive mutant (Glu1E191D) in complex with the natural substrate
2-
O
-β-
d
-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one
(DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor
para
-hydroxy-
S
-mandelonitrile
β-glucoside (dhurrin). The structures of these complexes and of the
free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-Å resolution,
respectively. The structural data from the complexes allowed us to
visualize an intact substrate, free aglycone, or a competitive
inhibitor in the slot-like active site of a β-glucosidase. These data
show that the aglycone moiety of the substrate is sandwiched between
W378 on one side and F198, F205, and F466 on the other. Thus, specific
conformations of these four hydrophobic amino acids and the shape of
the aglycone-binding site they form determine aglycone recognition and
substrate specificity in Glu1. In addition to these four residues, A467
interacts with the 7-methoxy group of DIMBOA. All residues but W378 are
variable among β-glucosidases that differ in substrate specificity,
supporting the conclusion that these sites are the basis of aglycone
recognition and binding (i.e., substrate specificity) in
β-glucosidases. The data also provide a plausible explanation for the
competitive binding of dhurrin to maize β-glucosidases with high
affinity without being hydrolyzed.
Maternal Child‐rearing Practices in Hong Kong and Beijing Chinese Families: A Comparative Study
Authors: Lai Alice Cheng , Zhang Zhi‐Xue , Wang Wen‐Zhong
Published: July 2002

The present study aimed to examine the child‐rearing practices in Chinese families in Beijing and Hong Kong. The sample consisted of 89 mothers in Beijing and 45 mothers in Hong Kong. The mothers were instructed to respond to the Child‐rearing Practice Report (CRPR) in Q‐sort format. Some of the item clusters were combined to produce the authoritarian and authoritative disciplinary styles. The results indicated that mothers in Hong Kong were more likely to adopt an authoritarian child‐rearing pattern than mothers in Beijing; however, the two groups did not differ in authoritative child‐rearing style. The results also showed that mothers in Hong Kong controlled their children more than their counterparts in Beijing, and they were less inclined to show affection towards their children; mothers in Beijing emphasized their children's achievement much more than their Hong Kong counterparts. The findings suggest that Chinese parental disciplinary styles may be quite different in various regions of Chinese societies. Such variance across different geopolitical locations within the same cultural background has been ignored in past cross‐cultural research.
Vietnamese Adolescents in Australia: Relationships between Perceptions of Self and Parental Values, Intergenerational Conflict, and Gender Dissatisfaction
Authors: Rosenthal Doreen , Ranieri Nadia , Klimidis Steven
Published: July 2002

The present study examined traditional family and independence values among young Vietnamese respondents (N = 204), their perceptions of parents' values, and the impact of differential parent‐adolescent acculturation on intergenerational conflict and gender satisfaction. The study confirmed that adolescents perceived that they had less traditional values than their parents. Traditional family values diminished with time spent in Australia while the value accorded to independence increased. This pattern was stronger for girls than for boys. Girls valued Vietnamese traditions less than did their male peers, regarded their parents as being less accepting of independence, and were more dissatisfied with their gender role than boys. For girls but not for boys, discrepancy between adolescent and parental values was associated with more conflict and greater gender dissatisfaction. This study suggests that girls have more difficulty than boys in dealing simultaneously with the expectations of two cultures.
An Economic Analysis of Convenience-store Location
Authors: Sakashita Noboru
Published: July 2002

This paper is concerned with convenience stores, and analyses the location (i.e. the distance between two neighbouring stores) and size of these stores, which are operated by one or two large retail companies. In order to maximise its profit, a company selects as its decision variables the distance between two neighbouring stores (or n, the number of its stores located within a given linear market area of fixed length N) and the size of an individual store. The outcomes of the monopolistic and duopolistic equilibria are compared with the 'socially optimal' outcome. Such solutions are dependent on the reservation price of the good held by consumers, the marginal supply cost of the good, the transport rate and the maintenance cost of an individual store. After the analytical presentation, certain numerical simulations are given, including a sensitivity analysis with regard to the transport rate. A large discrepancy between the equilibrium and the optimum is shown to exist.
Some Economic Extensions of Central-place Theory Involving Profit Maximisation
Authors: Ishikawa Toshiharu , Toda Masao
Published: July 2002

This paper attempts to extend central-place theory in terms of economic analysis, while at the same time refining and enhancing its applicability to urban systems. After reviewing the approach of Christaller, a central-place system is constructed, based on profit maximisation, and it is demonstrated that firms tend to concentrate at the largest central place. Next, the economic rationale for triangular market areas is discussed, and a central-place system is derived which involves the combination of triangular and hexagonal market areas. This model is used to explain the observed number of central places. Finally, the integration or separation of production and sales facilities is incorporated into the theory, as an attempt to explain the diversity of central places within an urban system.
The Local Housing System in Craigavon, N. Ireland: Ethno-religious Residential Segregation, Socio-tenurial Polarisation and Sub-markets
Authors: Adair A.S. , Berry J.N. , McGreal W.S.J. , Murtagh B. , Paris C.
Published: July 2002

The definition, identification and factors influencing the operation of local housing markets are relatively underresearched within the UK. This paper reports upon the outcomes of an investigation into the behaviour of housing markets within the local government area of Craigavon, Northern Ireland. The research investigated three interrelated themes of segregation, socio-tenurial polarisation and sub-markets. The findings indicate a high degree of ethno-religious segregation, clear evidence of socio-tenurial polarisation and the existence of sub-markets defined both by religion and locality. There is little evidence of mobility between the two main towns within the local government area, Portadown and Lurgan, but there is clear evidence of dual market structures within both towns.
UK Local Action Zones and Changing Urban Governance
Authors: Painter Chris , Clarence Emma
Published: July 2002

This article deals with UK Education and Health Action Zones, area-based partnership programmes, taking account of New Labour's 'modernising government' agenda and the academic partnership literature. Formative zone experiences again highlight major obstacles to effective local partnership working, exacerbated by the essentially top-down nature of the initiatives, albeit accommodating realities on the ground. Despite at one level prioritising changes in process, political pressures for rapid results were not easily compatible with policy innovation and longer-term collaborative infrastructure. Whether relating to the broader macro patterns of change affecting UK urban local governance to which action zones are contributing, or to the specific micro dynamics of these particular partnership initiatives, tensions inherent in the above dilemmas are far from being resolved.
Determinants of Population Growth in Urban Centres in the Republic of Ireland
Authors: Lutz James M.
Published: July 2002

Growth in urban places in Ireland from 1966 to 1991 has been greatly influenced by proximity to Dublin. In fact, the best predictor of growth has been such relative proximity. Government efforts to disperse growth to other areas of the Republic, however, have had some effects. Centres in the north-west and west of the country have grown more than would otherwise have been expected. While the growth in these areas may not be sustainable, the effects of previous government policies have been obvious.
Homo-Economicus in the City: Towards an Urban Socio-economic Research Agenda
Authors: Munro Moira
Published: July 2002

This paper seeks to outline the emerging debates and approaches in socio-economics, focusing particularly on the work of Etzioni. In relation to each of three core elements of the socio-economic agenda, possible applications to and implications for urban research are explored. Many of the ideas in the socio-economic approach require empirical testing and evaluation and it is argued that there are several aspects of the urban research agenda which provide plausible and potentially fertile grounds for applying the approach.