bluuebunny/crossref_metadata_2025 huggingface dataset:
Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence
Authors: Scheibel Thomas , Weikl Tina , Buchner Johannes
Published: July 2002

The abundant molecular chaperone Hsp90 is a key regulator of protein structure in the cytosol of eukaryotic cells. Although under physiological conditions a specific subset of proteins is substrate for Hsp90, under stress conditions Hsp90 seems to perform more general functions. However, the underlying mechanism of Hsp90 remained enigmatic. Here, we analyzed the function of conserved Hsp90 domains. We show that Hsp90 possesses two chaperone sites located in the N- and C-terminal fragments, respectively. The C-terminal fragment binds to partially folded proteins in an ATP-independent way potentially regulated by cochaperones. The N-terminal domain contains a peptide binding site that seems to bind preferentially peptides longer than 10 amino acids. Peptide dissociation is induced by ATP binding. Furthermore, the antitumor drug geldanamycin both inhibits the weak ATPase of Hsp90 and stimulates peptide release. We propose that the existence of two functionally different chaperone sites together with a substrate-selecting set of cochaperones allows Hsp90 to guide the folding of a subset of target proteins and, at the same time, to exhibit general chaperone functions.
Dynamics of contacts between lamellae of fibroblasts: Essential role of the actin cytoskeleton
Authors: Gloushankova N. A. , Krendel M. F. , Alieva N. O. , Bonder E. M. , Feder H. H. , Vasiliev J. M. , Gelfand I. M.
Published: July 2002

We investigated actin cytoskeletal and adhesion molecule dynamics during collisions of leading lamellae of nontransformed and oncogene-transformed fibroblasts. By using real-time video microscopy, it was found that during lamellar collision there was considerable overlapping of leading lamellae followed by subsequent retraction. Overlapping of nontransformed fibroblasts was accompanied by formation of β-catenin-positive contact structures organized into strands oriented parallel to the long axis of the cell that were associated with bundles of actin filaments. Maintenance of such cell–cell contact structures critically depended on the contractility of actin cytoskeleton, as inhibition of contractility with serum-free medium or 2,3-butanedione 2-monoxime (BDM) resulted in loss of strand formation. Strand formation was recovered when cells in serum-free medium were incubated with the microtubule inhibitor nocodazole, which is known to increase contractility. Oncogene-transformed fibroblasts reacted to collisions with responses similar to nontransformed fibroblasts but did not develop well-organized cell–cell contacts. A model is presented to describe how differences in the organization of the actin cytoskeleton could account for the structurally distinct responses to cell–cell contact by polarized fibroblastic cells versus nonpolarized epithelial cells.
Regulation of the zinc transporter ZnT-1 by dietary zinc
Authors: McMahon Robert J. , Cousins Robert J.
Published: July 2002

The understanding of mechanisms controlling zinc absorption and metabolism at the molecular level has advanced recently. Kinetics of zinc transport have been investigated for many years, but only recently have genes coding for proteins thought to be involved in the transport process been cloned. Four putative zinc transporters, known as ZnT-1 through ZnT-4, have now been described. Among these transporters, only ZnT-1 is ubiquitously expressed. In this report, we examine the pattern of ZnT-1 expression in the intestine and analyze the regulation of ZnT-1 by dietary zinc in both the intestine and liver. Immunofluorescence demonstrated that intestinal ZnT-1 was most abundant at the basolateral surface of enterocytes lining the villi of the duodenum and jejunum. By Western blot analysis, intestinal and liver ZnT-1 protein migrated as a 42- and 36-kDa protein, respectively. Dietary zinc supplementation elevated the level of intestinal ZnT-1 mRNA and protein approximately 50% and 10%, respectively, but had no effect in the liver. In response to an acute oral zinc dose, the level of intestinal ZnT-1 mRNA was up-regulated 8-fold, without a corresponding increase in ZnT-1 protein. Conversely, the acute oral dose did not affect liver ZnT-1 mRNA, but resulted in a 5-fold increase in liver ZnT-1 protein. These results represent studies on the expression of intestinal and hepatic ZnT-1 in an intact animal model. The data suggest that ZnT-1 is at least part of the mechanism by which dietary zinc is absorbed and that, despite the zinc responsiveness of the ZnT-1 gene, additional factors may be regulating the steady-state level of ZnT-1 transporter protein.
Heterophile antibodies segregate in families and are associated with protection from type 1 diabetes
Authors: She Jin-Xiong , Ellis Tamir M. , Wilson S. Brian , Wasserfall Clive H. , Marron Michele , Reimsneider Sharon , Kent Sally C. , Hafler David A. , Neuberg Donna S. , Muir Andy , Strominger Jack L. , Atkinson Mark A.
Published: July 2002

Markedly elevated levels of serum IL-4 were reported previously in 50% of a small group of type 1 diabetes nonprogessors. To determine the patterns of expression for this phenotype, a larger cohort of 58 families containing type 1 diabetic patients was examined. Analysis of the two-site ELISA assay used to measure serum IL-4 revealed evidence for heterophile antibodies, i.e., nonanalyte substances in serum capable of binding antibodies mutivalently and providing erroneous analyte (e.g., IL-4) quantification. Interestingly, relatives without type 1 diabetes were significantly more likely to have this phenotype than were patients with the disease (
P
= 0.003). In addition, the trait appears to have clustered within certain families and was associated with the protective MHC allele DQB1*0602 (
P
= 0.008). These results suggest that heterophile antibodies represent an
in vivo
trait associated with self-tolerance and nonprogression to diabetes.
Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy
Authors: Korlach Jonas , Schwille Petra , Webb Watt W. , Feigenson Gerald W.
Published: July 2002

We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chemically well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1,1′-dieicosanyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-C
20
) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-
s
-indacene-3-pentanoyl)-1-hexadecanoyl-
sn
-glycero-3-phosphocholine (Bodipy-PC). Phase separation was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal
z
-scans through giant unilamellar vesicles reveal the anisotropic morphology of coexisting phase domains on the surface of these vesicles with full two-dimensional resolution. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coefficient,
D
T
, of the DiI-C
20
was measured by FCS.
D
T
values at ∼25°C ranged from ∼ 3 × 10
−8
cm
2
/s in the fluid phase, to ∼ 2 × 10
−9
cm
2
/s in high-cholesterol-content phases, to ∼ 2 × 10
−10
cm
2
/s in the spatially ordered phases that coexist with fluid phases. In favorable cases, FCS could distinguish two different values of
D
T
in a region of two-phase coexistence on a single vesicle.
Structure of human factor VIIa and its implications for the triggering of blood coagulation
Authors: Pike Ashley C. W. , Brzozowski Andrzej M. , Roberts Shirley M. , Olsen Ole H. , Persson Egon
Published: July 2002

Factor VIIa (EC
3.4.21.21
) is a trypsin-like serine protease that plays a key role in the blood coagulation cascade. On injury, factor VIIa forms a complex with its allosteric regulator, tissue factor, and initiates blood clotting. Although the structure of the binary complex has already been determined [Banner, D. W., D’Arcy, A., Chène, C., Winkler, F. K., Guha, A., Konigsberg, W. H., Nemerson, Y. & Kirchhofer, D. (1996)
Nature (London)
380, 41–46], the conformational effects of cofactor binding to factor VIIa are not known in detail because of a lack of structural information on free factor VIIa. Here we report the structure of γ-carboxyglutamic acid-domainless human coagulation factor VIIa at a resolution of 2.8 Å. The molecule adopts an extended conformation within the crystal similar to that previously observed for the full-length protein in complex with tissue factor. Detailed comparison of free and tissue factor-bound factor VIIa reveals several structural differences. The binding mode of the active-site inhibitor
d
-Phe-Phe-Arg methyl ketone differs in the two structures, suggesting a role for the cofactor in substrate recognition. More importantly, a surface-exposed α-helix in the protease domain (residues 307–312), which is located at the cofactor recognition site, is distorted in the free form of factor VIIa. This subtle structural difference sheds light on the mechanism of the dramatic tissue factor-induced enhancement of factor VIIa activity.
Disruption of the murine nuclear factor I-A gene (
Nfia
) results in perinatal lethality, hydrocephalus, and agenesis of the corpus callosum
Authors: das Neves Liomar , Duchala Cynthia S. , Godinho Fatima , Haxhiu Musa A. , Colmenares Clemencia , Macklin Wendy B. , Campbell Christine E. , Butz Kenneth G. , Gronostajski Richard M.
Published: July 2002

The phylogenetically conserved nuclear factor I (NFI) family of transcription/replication proteins is essential both for adenoviral DNA replication and for the transcription of many cellular genes. We showed previously that the four murine NFI genes (
Nfia
,
Nfib
,
Nfic,
and
Nfix
) are expressed in unique but overlapping patterns during mouse development and in adult tissues. Here we show that disruption of the
Nfia
gene causes perinatal lethality, with >95% of homozygous
Nfia
−/−
animals dying within 2 weeks after birth. Newborn
Nfia
−/−
animals lack a corpus callosum and show ventricular dilation indicating early hydrocephalus. Rare surviving homozygous
Nfia
−/−
mice lack a corpus callosum, show severe communicating hydrocephalus, a full-axial tremor indicative of neurological defects, male-sterility, low female fertility, but near normal life spans. These findings indicate that while the
Nfia
gene appears nonessential for cell viability and DNA replication in embryonic stem cells and fibroblasts, loss of
Nfia
function causes severe developmental defects. This finding of an NFI gene required for a developmental process suggests that the four NFI genes may have distinct roles in vertebrate development.
Yersinia pestis
, the cause of plague, is a recently emerged clone of
Yersinia pseudotuberculosis
Authors: Achtman Mark , Zurth Kerstin , Morelli Giovanna , Torrea Gabriela , Guiyoule Annie , Carniel Elisabeth
Published: July 2002

Plague, one of the most devastating diseases of human history, is caused by
Yersinia pestis
. In this study, we analyzed the population genetic structure of
Y. pestis
and the two other pathogenic
Yersinia
species,
Y. pseudotuberculosis
and
Y. enterocolitica
. Fragments of five housekeeping genes and a gene involved in the synthesis of lipopolysaccharide were sequenced from 36 strains representing the global diversity of
Y. pestis
and from 12–13 strains from each of the other species. No sequence diversity was found in any
Y. pestis
gene, and these alleles were identical or nearly identical to alleles from
Y. pseudotuberculosis
. Thus,
Y. pestis
is a clone that evolved from
Y. pseudotuberculosis
1,500–20,000 years ago, shortly before the first known pandemics of human plague. Three biovars (Antiqua, Medievalis, and Orientalis) have been distinguished by microbiologists within the
Y. pestis
clone. These biovars form distinct branches of a phylogenetic tree based on restriction fragment length polymorphisms of the locations of the IS
100
insertion element. These data are consistent with previous inferences that Antiqua caused a plague pandemic in the sixth century, Medievalis caused the Black Death and subsequent epidemics during the second pandemic wave, and Orientalis caused the current plague pandemic.
The t(8;21) chromosomal translocation in acute myelogenous leukemia modifies intranuclear targeting of the AML1/CBFα2 transcription factor
Authors: McNeil Sandra , Zeng Congmei , Harrington Kimberly S. , Hiebert Scott , Lian Jane B. , Stein Janet L. , van Wijnen André J. , Stein Gary S.
Published: July 2002

Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1/ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1/ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias.
Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes
Authors: Beier Frank , Lee Richard J. , Taylor Allison C. , Pestell Richard G. , LuValle Phyllis
Published: July 2002

Endochondral bone growth is regulated by the rates of
chondrocyte proliferation and differentiation. However, the
intracellular mechanisms regulating these processes are poorly
understood. Recently, interruption of the gene encoding the
transcription factor activating transcription factor 2 (ATF-2) was
shown to inhibit proliferation of chondrocytes in mice [Reimold,
A. M.,
et al.
(1996)
Nature (London)
379, 262–265]. The target genes of ATF-2 that are responsible for
this phenotype remain unknown. Here we report that the cyclin D1 gene
is a direct target of ATF-2 in chondrocytes. ATF-2 is present in
nuclear extracts from chondrogenic cell lines and binds, as a complex
with a CRE-binding protein (CREB)/CRE modulator protein, to the cAMP
response element (CRE) in the cyclin D1 promoter. Mutation of the
cyclin D1 CRE caused a 78% reduction in the activity of the promoter
in chondrocytes. Overexpression of ATF-2 in chondrocytes enhanced
activity of the cyclin D1 promoter 3.5-fold. In contrast, inhibition of
endogenous ATF-2 or CREB by expression of dominant-negative
inhibitors of CREB and ATF-2 significantly reduced the activity of the
promoter in chondrocytes through the CRE. In addition, levels of cyclin
D1 protein are greatly reduced in the chondrocytes of ATF-2-deficient
mice. These data identify the cyclin D1 gene as a direct target of
ATF-2 in chondrocytes and suggest that reduced expression of cyclin D1
contributes to the defective cartilage development of these mice.
Transposon diversity in
Arabidopsis thaliana
Authors: Le Quang Hien , Wright Stephen , Yu Zhihui , Bureau Thomas
Published: July 2002

Recent availability of extensive genome sequence information offers new opportunities to analyze genome organization, including transposon diversity and accumulation, at a level of resolution that was previously unattainable. In this report, we used sequence similarity search and analysis protocols to perform a fine-scale analysis of a large sample (≈17.2 Mb) of the
Arabidopsis thaliana
(Columbia) genome for transposons. Consistent with previous studies, we report that the
A. thaliana
genome harbors diverse representatives of most known superfamilies of transposons. However, our survey reveals a higher density of transposons of which over one-fourth could be classified into a single novel transposon family designated as
Basho
, which appears unrelated to any previously known superfamily. We have also identified putative transposase-coding ORFs for miniature inverted-repeat transposable elements (MITEs), providing clues into the mechanism of mobility and origins of the most abundant transposons associated with plant genes. In addition, we provide evidence that most mined transposons have a clear distribution preference for A + T-rich sequences and show that structural variation for many mined transposons is partly due to interelement recombination. Taken together, these findings further underscore the complexity of transposons within the compact genome of
A. thaliana
.
Loss of
BETA2/NeuroD
leads to malformation of the dentate gyrus and epilepsy
Authors: Liu Min , Pleasure Samuel J. , Collins Abigail E. , Noebels Jeffrey L. , Naya Francesco J. , Tsai Ming-Jer , Lowenstein Daniel H.
Published: July 2002

BETA2/NeuroD
is a homologue of the
Drosophila
atonal gene that is widely expressed during development in the mammalian brain and pancreas. Although studies in
Xenopus
suggest that
BETA2/NeuroD
is involved in cellular differentiation, its function in the mammalian nervous system is unclear. Here we show that mutant mice homozygous for a deletion at the
BETA2/NeuroD
locus fail to develop a granule cell layer within the dentate gyrus, one of the principal structures of the hippocampal formation. To understand the basis of this abnormality, we analyzed dentate gyrus development by using immunocytochemical markers in
BETA2/NeuroD
-deficient mice. The early cell populations in the dentate gyrus, including Cajal–Retzius cells and radial glia, are present and appear normally organized. The migration of dentate precursor cells and newly born granule cells from the neuroepithelium to the dentate gyrus remains intact. However, there is a dramatic defect in the proliferation of precursor cells once they reach the dentate and a significant delay in the differentiation of granule cells. This leads to malformation of the dentate granule cell layer and excess cell death.
BETA2/NeuroD
null mice also exhibit spontaneous limbic seizures associated with electrophysiological evidence of seizure activity in the hippocampus and cortex. These findings thus establish a critical role of
BETA2/NeuroD
in the development of a specific class of neurons. Furthermore, failure to express
BETA2/NeuroD
leads to a stereotyped pattern of pathological excitability of the adult central nervous system.
Predicting species diversity in tropical forests
Authors: Plotkin Joshua B. , Potts Matthew D. , Yu Douglas W. , Bunyavejchewin Sarayudh , Condit Richard , Foster Robin , Hubbell Stephen , LaFrankie James , Manokaran N. , Seng Lee Hua , Sukumar Raman , Nowak Martin A. , Ashton Peter S.
Published: July 2002

A fundamental question in ecology is how many species occur within
a given area. Despite the complexity and diversity of different
ecosystems, there exists a surprisingly simple, approximate answer: the
number of species is proportional to the size of the area raised to
some exponent. The exponent often turns out to be roughly 1/4. This
power law can be derived from assumptions about the relative abundances
of species or from notions of self-similarity. Here we analyze the
largest existing data set of location-mapped species: over one million,
individually identified trees from five tropical forests on three
continents. Although the power law is a reasonable, zeroth-order
approximation of our data, we find consistent deviations from it on all
spatial scales. Furthermore, tropical forests are not self-similar at
areas ≤50 hectares. We develop an extended model of the
species-area relationship, which enables us to predict large-scale
species diversity from small-scale data samples more accurately than
any other available method.
Memory's echo: Vivid remembering reactivates sensory-specific cortex
Authors: Wheeler Mark E. , Petersen Steven E. , Buckner Randy L.
Published: July 2002

A fundamental question in human memory is how the brain represents
sensory-specific information during the process of retrieval. One
hypothesis is that regions of sensory cortex are reactivated during
retrieval of sensory-specific information (1). Here we report
findings from a study in which subjects learned a set of picture and
sound items and were then given a recall test during which they vividly
remembered the items while imaged by using event-related functional
MRI. Regions of visual and auditory cortex were activated
differentially during retrieval of pictures and sounds, respectively.
Furthermore, the regions activated during the recall test comprised a
subset of those activated during a separate perception task in which
subjects actually viewed pictures and heard sounds. Regions activated
during the recall test were found to be represented more in late than
in early visual and auditory cortex. Therefore, results indicate that
retrieval of vivid visual and auditory information can be associated
with a reactivation of some of the same sensory regions that were
activated during perception of those items.
Recombination within natural populations of pathogenic bacteria: Short-term empirical estimates and long-term phylogenetic consequences
Authors: Feil Edward J. , Holmes Edward C. , Bessen Debra E. , Chan Man-Suen , Day Nicholas P. J. , Enright Mark C. , Goldstein Richard , Hood Derek W. , Kalia Awdhesh , Moore Catrin E. , Zhou Jiaji , Spratt Brian G.
Published: July 2002

The identification of clones within bacterial populations is often
taken as evidence for a low rate of recombination, but the validity of
this inference is rarely examined. We have used statistical tests of
congruence between gene trees to examine the extent and significance of
recombination in six bacterial pathogens. For
Neisseria
meningitidis
,
Streptococcus pneumoniae
,
Streptococcus pyogenes,
and
Staphylococcus
aureus
, the congruence between the maximum likelihood trees
reconstructed using seven house-keeping genes was in most cases no
better than that between each tree and trees of random topology. The
lack of congruence between gene trees in these four species, which
include both naturally transformable and nontransformable species, is
in three cases supported by high ratios of recombination to point
mutation during clonal diversification (estimates of this parameter
were not possible for
Strep. pyogenes
). In contrast,
gene trees constructed for
Hemophilus influenzae
and
pathogenic isolates of
Escherichia coli
showed a higher
degree of congruence, suggesting lower rates of recombination. The
impact of recombination therefore varies between bacterial species but
in many species is sufficient to obliterate the phylogenetic signal in
gene trees.
Engineering of a functional human NADH-dependent cytochrome P450 system
Authors: Döhr Olaf , Paine Mark J. I. , Friedberg Thomas , Roberts Gordon C. K. , Wolf C. Roland
Published: July 2002

A functional human NADH-dependent cytochrome P450 system has been
developed by altering the cofactor preference of human NADPH cytochrome
P450 reductase (CPR), the redox partner for P450s. This has been
achieved by a single amino acid change of the conserved aromatic amino
acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in
the nicotinamide-binding site. Of the mutations made, the substitution
of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent
enzyme, which catalyzed the reduction of cytochrome
c
and ferricyanide as well as facilitated the metabolism of
7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome
c
activity revealed that the NADH-dependent
k
cat
of W676A is equivalent (90%) to the
NADPH-dependent
k
cat
of the wild-type
enzyme, with W676A having an approximately 1,000-fold higher
specificity for NADH. The apparent
K
M
NADPH
and
K
M
NADH
values of W676A are 80- and
150-fold decreased, respectively. In accordance with structural data,
which show a bipartite binding mode of NADPH, substitution of Trp-676
does not affect 2′-AMP binding as seen by the inhibition of both
wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent
inhibitor of the W676A NADH-dependent cytochrome
c
reduction and CYP1A2 activity. Overall, the results show that Trp-676
of human CPR plays a major role in cofactor discrimination, and
substitution of this conserved aromatic residue with alanine results in
an efficient NADH-dependent cytochrome P450 system.
Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans
Authors: Ohno Kinji , Tsujino Akira , Brengman Joan M. , Harper C. Michel , Bajzer Zeljko , Udd Bjarne , Beyring Roger , Robb Stephanie , Kirkham Fenella J. , Engel Andrew G.
Published: July 2002

Choline acetyltransferase (ChAT; EC2.3.1.6) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist inCaenorhabditis elegansandDrosophila, but noCHATmutations have been observed in humans to date. Here we report that mutations inCHATcause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and toCHATas one of the candidate genes. Direct sequencing ofCHATreveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.
Local Economic Development Strategies in an Emerging Democracy: The Case of Durban in South Africa
Authors: Maharaj Brij , Ramballi Kem
Published: July 2002

In recent years, especially since 1990, there has been an increased emphasis on LED strategies as South African cities focus on urban reconstruction and development to try to reduce the inequalities associated with apartheid. The aim of this paper is to investigate the nature of local economic development strategies in Durban, with specific reference to the Point Redevelopment Programme and the International Convention Centre. It was widely expected that these projects would create jobs and promote tourism. These projects were initially largely driven by business and local state interests, and there was no public participation. Pressure from community groups led to a fundamental restructuring of the development process. The formation of the Durban Infrastructural Development Trust was a progressive strategy to get the wider community involved in the process. The Trust released a protocol which stipulated that the guiding principles for these projects would be widespread consultation in the planning and development process, as well as policies for job-creation and affirmative action. However, this paper questions whether these objectives will be realised, and whether the disadvantaged communities will benefit from the local economic development projects. An analysis of the financing of the projects and concessions granted to private developers by the local state reveals that most of the funding would come from the tax-payer. Therefore, inequalities would not be addressed and any benefits to the community would only take place by the trickle-down effect.
Muslim Roma in the Balkans
Authors: Crowe David M.
Published: July 2002

The Roma entered the Balkans from India during the Middle Ages. They reached Persia sometime in the ninth century and by the eleventh century had moved into the Byzantine Empire. According to the eleventh-century GeorgianLife of Saint George the Athonite, the Emperor Constantine Monomachus asked theAdsincanito get rid of wild animals preying on the animals in his royal hunting preserve.Adsincaniis the Georgian form of the Greek wordAtsínganoiorAtzínganoi, from which the non-English terms for Roma (cigán, cigány, tsiganes, zigeuner) are derived.Adsincanimeans “ner-do-well fortune tellers” or “ventriloquists and wizards who are inspired satanically and pretend to predict the unknown.” “Gypsy” comes from “Egyptian,” a term often used by early modern chroniclers in the Balkans to refer to the Roma. Because of the stereotypes and prejudice that surround the word “Gypsy,” the Roma prefer a name of their own choosing from their language, Romani. Today, it is preferable to refer to the Gypsies asRomor “Roma,” a Romani word meaning “man” or “husband.” Byzantine references to “Egyptians” crop up during this period as Byzantine political and territorial fortunes gave way to the region's new power, the Ottomans. There were areas with large Roma populations in Cyprus and Greece which local rulers dubbed “Little Egypt” in the late fourteenth century.
Pomak Borderlands: Muslims on the Edge of Nations
Authors: Neuburger Mary
Published: July 2002

We contact the worldonly through our boundaries.Blaga Dimitrova

In a recent issue of the Bulgarian periodicalSega(Now) a reporter related an extraordinary tale of how various name-changing campaigns had marked the experience of a Bulgarian-speaking Muslim—hereafter “Pomak”—in the village of Bachkovo. The story began during the Balkan Wars in 1912–1913 when Hasan, the aforementioned Pomak from the Rhodope mountains of southern Bulgaria, was forced to change his name to Dragan as part of the wartime state campaign for Muslims with “Slavic origins” to “reclaim their Bulgarian names.” A change in politics at the beginning of World War I opened the door for Dragan to change his name back to Hasan; and so he did. In the late 1930s, however, he was again compelled to change his name back to Dragan, in line with theRodina(Homeland) directed name-changing campaigns, described in depth below. After the Communist takeover in 1944 Dragan was able, again, to change his name back to Hasan as wartime “Fascist” policy was reversed. But with the movement towards “national integration” in the 1960s Hasan was forced, again, to change his name back to Dragan. After the fall of Communism in Bulgaria in November 1989 “Dragan” again was allowed to change his name back to Hasan; and so he did. In his one lifetime this “Bulgarian” of Islamic faith, subject to the whims of the fickle and contested Bulgarian national project, changed his name six times. Admittedly, the Pomak's fate in Balkan history seems to be primarily as pawn in Bulgarian and other Balkan national rivalries and domestic designs. Pomak history is, more often than not, the story of the center looking to the margins and imposing its own designs. Having said that, these designs—generally driven by the dual forces of modernity and nationalism—were always subject to a spectrum of Pomak responses and strategies.
City-regional Systems and Competitive Internet Economies
Authors: McCready Philip
Published: July 2002

A live issue currently in eonomic development relates to the importance of city-regional business environments for the growth and competitiveness of internet-related industries. Drawing on theories of regional innovation systems, this study evaluates the importance of place-related factors in the development of internet firms in two UK city-regions. The findings highlight the importance of demand economies, external networks, key local firms and individuals, and government research expenditure. Institutional arguments about local-regional systems are not drivers for growth, but play a more complex role in shaping knowledge and technological infrastructure.
Genitive Modifiers, Sorts, and Metonymy
Authors: Borschev Vladimir , Partee Barbara H.
Published: July 2002

Our long-term goal is to contribute to the integration of formal and lexical semantics. Our more immediate theoretical starting point is the idea of “text as theory”, within a model-theoretic semantic framework. We describe a set of empirical problems in the domain of genitive modifiers that offers a challenge to theories of the integration of lexical, compositional, and contextual information. After sketching a solution, we raise the issue of metonymy in the interpretation of genitives, and examine the role of sortal information in the specification of underspecified meanings and in processes of type-shifting and sort-shifting, including metonymy.
Failure of 5‐HT3receptors in regulation of ethanol‐induced ascorbic acid release in rat striatum
Authors: Wu Chun‐Fu , Liu Jing , Liu Wen , Consolo Silvana , Huang Mei , Yang Jing‐Yu
Published: July 2002

Previous studies have shown that the serotonergic system was involved in ethanol‐induced striatal ascorbic acid release in rats. In the present study the possible role of 5‐HT 3 receptors in ethanol‐induced striatal ascorbic acid release was investigated in rats using 5‐HT 3 antagonists ondansetron, DAU 6215 and 5‐HT 3 agonist 2‐methyl‐serotonin. Extracellular level of ascorbic acid in the striatum was determined by means of in vivo microdialysis coupled to HPLC with electrochemical detection. Ethanol (3 g/kg, i.p.) induced a significant increase in ascorbic acid release. Ondansetron (0.2 and 2.0 mg/kg, i.p.), DAU 6215 (0.06, 0.12 and 0.24 mg/kg, i.p.) and 2‐methyl‐serotonin (250 μg/rat, i.c.v.), administered 10 minutes before 0.15 M NaCl or ethanol (3 g/kg, i.p.), affect neither the basal nor the ethanol‐induced ascorbic acid release in rat striatum. 2‐Methylserotonin, at a dose of 500 μg/rat, i.c.v., increased the basal, but did not affect the ethanol‐induced ascorbic acid release in rat striatum. However, ritanserin (1 mg/kg, s.c.), a 5‐HT 2 receptor antagonist, and BIMU 8 (40 μg/rat, i.c.v.), a 5‐HT 4 agonist, significantly antagonized ethanol‐induced ascorbic acid release. These results suggest that 5‐HT 3 receptors, which form a part of cation channels, may not be involved in ethanolinduced striatal ascorbic acid release.
Perception of aspiration and place of articulation of Cantonese initial stops by normal and sensorineural hearing‐impaired listeners
Authors: Y. H. Tsui Ida , Ciocca Valter
Published: July 2002

This study investigated the use of acoustic cues to the perception of aspiration and place of articulation of Cantonese initial stops by bilateral severely hearingimpaired (n =14) and normal‐hearing (n =14) adolescents. The stimuli were consonant‐vowel (CV) words spoken by two male speakers in which the initial consonants /p h, p, t h, t, k h, k/ were followed by the diphthong /i/. Subjects listened to the stimuli through a loudspeaker and chose the correct initial consonant among six choices. Three test conditions (short, medium and long voice onset time, or VOT) were prepared by increasing the VOT of unaspirated stops and by decreasing the VOT of aspirated stops. The results showed that the presence of aspiration noise was an important cue for normal listeners in the perception of aspiration, but not for hearing‐impaired listeners. Hearingimpaired listeners used formant transitions as the main cue to the perception of aspiration. VOT was a weak aspiration cue for both groups. In the perception of place of articulation, normal listeners appeared to rely mainly on formant transitions and release burst information rather than VOT. The recognition of place by hearing‐impaired listeners was at chance level for all the experimental stimuli, showing that these listeners were unable to use VOT, formant transitions or release burst information as cues to place.
Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail
Authors: Kashiwagi Hirokazu , Schwartz Martin A. , Eigenthaler Martin , Davis K.A. , Ginsberg Mark H. , Shattil Sanford J.
Published: July 2002

Platelet agonists increase the affinity state of integrin αIIbβ3, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β3-Endonexin is a novel 111–amino acid protein that binds selectively to the β3 tail. Since β3-endonexin is present in platelets, we asked whether it can affect αIIbβ3 function. When β3-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor αIIbβ3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and αIIbβ3 bound little or no PAC1. However, those transfected with GFP/β3-endonexin and αIIbβ3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β3-endonexin did not affect levels of surface expression of αIIbβ3 nor did it modulate the affinity of an αIIbβ3 mutant that is defective in binding to β3-endonexin. Affinity modulation of αIIbβ3 by GFP/β3-endonexin was inhibited by coexpression of either a monomeric β3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β3-endonexin can modulate the affinity state of αIIbβ3 in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of αIIbβ3.
The Role of ADP-ribosylation Factor and Phospholipase D in Adaptor Recruitment
Authors: West Michele A. , Bright Nicholas A. , Robinson Margaret S.
Published: July 2002

AP-1 and AP-2 adaptors are recruited onto the TGN and plasma membrane, respectively. GTPγS stimulates the recruitment of AP-1 onto the TGN but causes AP-2 to bind to an endosomal compartment (Seaman, M.N.J., C.L. Ball, and M.S. Robinson. 1993. J. Cell Biol. 123:1093–1105). We have used subcellular fractionation followed by Western blotting, as well as immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to endosomes, and we have also examined the recruitment of AP-1 under the same conditions. Two lines of evidence indicate that the GTPγS-induced targeting of AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTPγS loses its effect when added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding GTPγS. The endosomal membranes that recruit AP-2 adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting catalytically. The ARFs have been shown to activate phospholipase D (PLD), and we find that addition of exogenous PLD has the same effect as GTPγS or Q71L ARF1. Neomycin, which inhibits endogenous PLD by binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the TGN, even though AP-1 recruitment is ARF mediated. These results indicate that different mechanisms are used for the recruitment of AP-1 and AP-2.
Specific and Redundant Functions of Retinoid X Receptor/Retinoic Acid Receptor Heterodimers in Differentiation, Proliferation, and Apoptosis of F9 Embryonal Carcinoma Cells
Authors: Chiba Hideki , Clifford John , Metzger Daniel , Chambon Pierre
Published: July 2002

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)α and retinoic acid receptor (RAR)α genes or the RXRα and RARγ genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRα−/−, RARα−/−, and RARγ−/− cells. RXRα−/−/ RARα−/− cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRα−/− cells. In contrast, RXRα−/−/RARγ−/− cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRα and RARγ represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype– and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.
Stimulation of NSF ATPase Activity by α-SNAP Is Required for SNARE Complex Disassembly and Exocytosis
Authors: Barnard Richard J.O. , Morgan Alan , Burgoyne Robert D.
Published: July 2002

N-ethylmaleimide–sensitive fusion protein (NSF) and α-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH2- and COOH-terminal truncation mutants of α-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids had little effect on the ability of α-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease. Both NH2-terminal (1–160) and COOH-terminal (160–295) fragments of α-SNAP were able to bind to NSF, suggesting that α-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of α-SNAP. Mutation of leucine 294 to alanine (α-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. α-SNAP (1–285) and α-SNAP (L294A) were unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, α-SNAP (1–285), and α-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous α-SNAP. α-SNAP, α-SNAP (1–285), and α-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, α-SNAP (1–285) and α-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that α-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the α-SNAP stimulation of exocytosis.
Molecular Motors and a Spectrin Matrix Associate with Golgi Membranes In Vitro
Authors: Fath Karl R. , Trimbur Gina M. , Burgess David R.
Published: July 2002

Cytoplasmic dynein is a microtubule minus-end–directed motor that is thought to power the transport of vesicles from the TGN to the apical cortex in polarized epithelial cells. Trans-Golgi enriched membranes, which were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isolated Golgi stacks lack dynein but contain myosin-I (Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. J. Cell Biol. 126:661–675). We show now that Golgi stacks in vitro bind dynein supplied from cytosol in the absence of ATP, and bud small membranes when incubated with cytosol and ATP. Cytosolic dynein binds to regions of stacks that are destined to bud because dynein is present in budded membranes, but absent from stacks after budding. Budded membranes move exclusively towards microtubule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resists extraction by ice-cold Triton X-100. In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.
Apical Plasma Membrane Proteins and Endolyn-78 Travel through a Subapical Compartment in Polarized WIF-B Hepatocytes
Authors: Ihrke Gudrun , Martin Greg V. , Shanks Michael R. , Schrader Michael , Schroer Trina A. , Hubbard Ann L.
Published: July 2002

We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37°C by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5′nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti–endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.