bluuebunny/crossref_metadata_2025 huggingface dataset:
Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model
Authors: Greenstone Heather L. , Nieland John D. , de Visser Karin E. , De Bruijn Marloes L. H. , Kirnbauer Reinhard , Roden Richard B. S. , Lowy Douglas R. , Kast W. Martin , Schiller John T.
Published: July 2002

Papillomavirus-like particles (VLPs) are a promising prophylactic vaccine candidate to prevent human papillomavirus (HPV) infections and associated epithelial neoplasia. However, they are unlikely to have therapeutic effects because the virion capsid proteins are not detected in the proliferating cells of the infected epithelia or in cervical carcinomas. To increase the number of viral antigen targets for cell-mediated immune responses in a VLP-based vaccine, we have generated stable chimeric VLPs consisting of the L1 major capsid protein plus the entire E7 (11 kDa) or E2 (43 kDa) nonstructural papillomavirus protein fused to the L2 minor capsid protein. The chimeric VLPs are indistinguishable from the parental VLPs in their morphology and in their ability to agglutinate erythrocytes and elicit high titers of neutralizing antibodies. Protection from tumor challenge was tested in C57BL/6 mice by using the tumor cell line TC-1, which expresses HPV16 E7, but not the virion structural proteins. Injection of HPV16 L1/L2-HPV16 E7 chimeric VLPs, but not HPV16 L1/L2 VLPs, protected the mice from tumor challenge, even in the absence of adjuvant. The chimeric VLPs also induced protection against tumor challenge in major histocompatibility class II-deficient mice, but not in β2-microglobulin or perforin knockout mice implying that protection was mediated by class I-restricted cytotoxic lymphocytes. These findings raise the possibility that VLPs may generally be efficient vehicles for generating cell-mediated immune responses and that, specifically, chimeric VLPs containing papillomavirus nonstructural proteins may increase the therapeutic potential of VLP-based prophylactic vaccines in humans.
Cloning and characterization of a novel endothelin receptor subtype in the avian class
Authors: Lecoin Laure , Sakurai Takeshi , Ngo Minh-Triet , Abe Yoichiro , Yanagisawa Masashi , Le Douarin Nicole M.
Published: July 2002

Endothelin 3 (EDN 3) and the endothelin receptor B (EDNRB) are involved in the development of neural crest and particularly of the melanocytes and the enteric nervous system. We reported previously that the avian
EDNRB
gene is expressed in the neural fold before crest cell migration and later on in all the neural crest derivatives except, at any developmental stage, in the melanocytic lineage. However, quail melanoblasts proliferate in response to EDN 3 stimulation
in vitro
. These observations prompted us to search for another type of endothelin receptor (EDNR). We report here the cloning by reverse transcriptase–PCR of an avian cDNA encoding a subtype of EDNR, which we have called EDNRB2, because its deduced amino acid sequence is more closely related to that of EDNRB than to either the mammalian EDNRA or to the
Xenopus
EDNRC. Its expression pattern differs from that of the “classical” avian
EDNRB
because it is strongly expressed in melanoblasts and melanocytes.
EDNRB2
transcripts are also abundant in the liver and kidney. Our pharmacological studies showed that EDNRB2 binds with similar affinity to EDN 1, EDN 2, and EDN 3, further confirming that this receptor belongs to the B type, although it displays a low affinity for sarafotoxin-c, a known EDNRB-selective agonist.
Alignment enhances the cell-to-cell transfer of pilus phenotype
Authors: Wall Daniel , Kaiser Dale
Published: July 2002

Social gliding motility of
Myxococcus xanthus
requires polar type IV pili. Tgl mutants lack pili and lack social motility. However, both defects can be rescued phenotypically, but not genotypically, when
tgl
+
donor and
tgl
−
recipient cells make physical contact with each other. What is the cellular and molecular basis of this transfer of phenotype, which is called stimulation? Stimulation does not occur in liquid nor in soft (0.5%) agar; however, on a more firm surface (1.0% agar) cells stimulate each other efficiently. Microscopy revealed that cells placed on 1.0% agar readily became aligned whereas they remained poorly aligned on 0.5% agar. It was observed, moreover, that stimulation is greatly reduced when donor and recipient cells lack the ability to move because of mutations in any of several different motility genes. Microscopy showed that motile cells became highly aligned, and can even align nonmotile cells. Using a stimulation assay based on the assembly of pili, a strong correlation was found between conditions that promote cell alignment and the speed or extent of stimulation. Because pili are assembled only at the end(s) of a cell, and a parallel alignment of cells in contact brings them end-to-end, we would suggest that end-to-end contacts are important for stimulation of pilus assembly.
Gene number in an invertebrate chordate,
Ciona intestinalis
Authors: Simmen Martin W. , Leitgeb Sabine , Clark Victoria H. , Jones Steven J. M. , Bird Adrian
Published: July 2002

Gene number can be considered a pragmatic measure of biological complexity, but reliable data is scarce. Estimates for vertebrates are 50-100,000 genes per haploid genome, whereas invertebrate estimates fall below 25,000. We wished to test the hypothesis that the origin of vertebrates coincided with extensive gene creation. A prediction is that gene number will differ sharply between invertebrate and vertebrate members of the chordate phylum. A gene number estimation method requiring limited sequence sampling of genomic DNA was developed and validated by using data for
Caenorhabditis elegans
. Using the method, we estimated that the invertebrate chordate
Ciona intestinalis
has 15,500 protein-coding genes (±3,700). This number is significantly lower than gene numbers of vertebrate chordates, but similar to those of invertebrates in distantly related phyla. The data indicate that evolution of vertebrates was accompanied by a dramatic increase in protein-coding capacity of the genome.
Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements
Authors: D’Souza Ian , Poorkaj Parvoneh , Hong Ming , Nochlin David , Lee Virginia M.-Y. , Bird Thomas D. , Schellenberg Gerard D.
Published: July 2002

Frontotemporal dementia with parkinsonism, chromosome 17 type (FTDP-17) is caused by mutations in the tau gene, and the signature lesions of FTDP-17 are filamentous tau inclusions. Tau mutations may be pathogenic either by altering protein function or gene regulation. Here we show that missense, silent, and intronic tau mutations can increase or decrease splicing of tau exon 10 (E10) by acting on 3 different cis-acting regulatory elements. These elements include an exon splicing enhancer that can either be strengthened (mutation
N
279
K
) or destroyed (mutation Δ280
K
), resulting in either constitutive E10 inclusion or the exclusion of E10 from tau transcripts. E10 contains a second regulatory element that is an exon splicing silencer, the function of which is abolished by a silent FTDP-17 mutation (
L
284
L
), resulting in excess E10 inclusion. A third element inhibiting E10 splicing is contained in the intronic sequences directly flanking the 5′ splice site of E10 and intronic FTDP-17 mutations in this element enhance E10 inclusion. Thus, tau mutations cause FTDP-17 by multiple pathological mechanisms, which may explain the phenotypic heterogeneity observed in FTDP-17, as exemplified by an unusual family described here with tau pathology as well as amyloid and neuritic plaques.
An aminoacyl-tRNA synthetase paralog with a catalytic role in histidine biosynthesis
Authors: Sissler Marie , Delorme Christine , Bond Jeff , Ehrlich S. Dusko , Renault Pierre , Francklyn Christopher
Published: July 2002

In addition to their essential catalytic role in protein biosynthesis, aminoacyl-tRNA synthetases participate in numerous other functions, including regulation of gene expression and amino acid biosynthesis via transamidation pathways. Herein, we describe a class of aminoacyl-tRNA synthetase-like (HisZ) proteins based on the catalytic core of the contemporary class II histidyl-tRNA synthetase whose members lack aminoacylation activity but are instead essential components of the first enzyme in histidine biosynthesis ATP phosphoribosyltransferase (HisG). Prediction of the function of HisZ in
Lactococcus lactis
was assisted by comparative genomics, a technique that revealed a link between the presence or the absence of HisZ and a systematic variation in the length of the HisG polypeptide. HisZ is required for histidine prototrophy, and three other lines of evidence support the direct involvement of HisZ in the transferase function. (
i
) Genetic experiments demonstrate that complementation of an in-frame deletion of HisG from
Escherichia coli
(which does not possess HisZ) requires both HisG and HisZ from
L. lactis
. (
ii
) Coelution of HisG and HisZ during affinity chromatography provides evidence of direct physical interaction. (
iii
) Both HisG and HisZ are required for catalysis of the ATP phosphoribosyltransferase reaction. This observation of a common protein domain linking amino acid biosynthesis and protein synthesis implies an early connection between the biosynthesis of amino acids and proteins.
Biochemical analysis of a dimerization domain mutation in RetGC-1 associated with dominant cone–rod dystrophy
Authors: Tucker Chandra L. , Woodcock Sarah C. , Kelsell Rosemary E. , Ramamurthy Visvanathan , Hunt David M. , Hurley James B.
Published: July 2002

Mutations in the photoreceptor membrane guanylyl cyclase RetGC-1 have been linked to autosomal dominant cone–rod dystrophy. Three mutations were identified that alter strictly conserved residues within the RetGC-1 dimerization domain, a region predicted to form an amphipathic α-helical coil. Here we report on a biochemical characterization of one of the mutations, a substitution of cysteine for arginine at residue 838. We generated this mutation
in vitro
and measured its catalytic activity and sensitivity to guanylyl cyclase activating protein 1 (GCAP-1) and GCAP-2. The R838C substitution has several effects. It reduces the overall catalytic ability of RetGC-1 and dramatically reduces stimulation by GCAP-2, although GCAP-2 still appears to interact with the protein. The R838C substitution also increases the apparent affinity of RetGC-1 for GCAP-1 and alters the Ca
2+
sensitivity of the GCAP-1 response, allowing the mutant to be stimulated by GCAP-1 at higher Ca
2+
concentrations than wild type. The diminished response to GCAP-2, which we propose is not likely the cause of cone–rod degeneration in these patients, is interesting mechanistically because it separates the ability to bind a specific GCAP from the ability to be stimulated by it, and it also discriminates between the mechanisms of activation of GCAP-1 vs. GCAP-2. We suggest that the gain-of-function effects of R838C on RetGC-1 stimulated by GCAP-1, which are dominant
in vitro
and may cause an abnormal increase in cGMP synthesis in dark-adapted photoreceptors, may be the cause of the cone–rod degeneration.
Receptor-mediated uptake of an extracellular Bcl-xLfusion protein inhibits apoptosis
Authors: Liu Xiu-Huai , Castelli JoAnn C. , Youle Richard J.
Published: July 2002

Bcl-xL, a member of the Bcl-2 family, inhibits many pathways of apoptosis when overexpressed in the cell cytosol. We examined the capacity of Bcl-xLfusion proteins to bind cells from the outside and block apoptosis. Full-length Bcl-xLprotein at micromolar concentrations did not affect apoptosis when added to cell media. To increase uptake by cells, Bcl-xLwas fused to the receptor-binding domain of diphtheria toxin (DTR). The Bcl-xL–DTR fusion protein blocked apoptosis induced by staurosporine, γ-irradiation, and poliovirus in a variety of cell types when added to media. The potency of inhibition of poliovirus-induced apoptosis by Bcl-xL–DTR was greater than that of strong caspase inhibitors. Brefeldin A, an inhibitor of vesicular traffic between the endoplasmic reticulum and Golgi apparatus, prevented the Bcl-xL–DTR blockade of apoptosis induced by staurosporine, suggesting that Bcl-xL–DTR must be endocytosed and reach intracellular compartments for activity. Many diseases are caused by overexpression or underexpression of Bcl-xLhomologues. Extracellular delivery of Bcl-2 family member proteins may have a wide range of uses in promoting or preventing cell death.
The speed limit for protein folding measured by triplet–triplet energy transfer
Authors: Bieri Oliver , Wirz Jakob , Hellrung Bruno , Schutkowski Mike , Drewello Mario , Kiefhaber Thomas
Published: July 2002

A direct measure of intramolecular chain diffusion is obtained by the determination of triplet–triplet energy-transfer rates between a donor and an acceptor chromophore attached at defined points on a polypeptide chain. Single exponential kinetics of contact formation are observed on the nanosecond time scale for polypeptides in which donor and acceptor are linked by repeating units of glycine and serine residues. The rates depend on the number of peptide bonds (
N
) separating donor and acceptor and show a maximum for the shortest peptides (
N
= 3) with a time constant (
τ
= 1/
k
) of 20 ns. This sets an upper limit for the speed of formation of the first side-chain contacts during protein folding.
Impaired glucose homeostasis and neonatal mortality in hepatocyte nuclear factor 3α-deficient mice
Authors: Shih David Q. , Navas M. Angeles , Kuwajima Satoru , Duncan Stephen A. , Stoffel Markus
Published: July 2002

Hepatocyte nuclear factors 3 (HNF-3) belong to an evolutionarily conserved family of transcription factors that are critical for diverse biological processes such as development, differentiation, and metabolism. To study the physiological role of HNF-3α, we generated mice that lack HNF-3α by homologous recombination in embryonic stem cells. Mice homozygous for a null mutation in the HNF-3α gene develop a complex phenotype that is characterized by abnormal feeding behavior, progressive starvation, persistent hypoglycemia, hypotriglyceridemia, wasting, and neonatal mortality between days 2 and 14. Hypoglycemia in HNF-3α-null mice leads to physiological counter-regulatory responses in glucocorticoid and growth hormone production and an inhibition of insulin secretion but fails to stimulate glucagon secretion. Glucagon-producing pancreatic alpha cells develop normally in HNF-3α−/− mice, but proglucagon mRNA levels are reduced 50%. Furthermore, the transcriptional levels of neuropeptide Y are also significantly reduced shortly after birth, implying a direct role of HNF-3α in the expression of these genes. In contrast, mRNA levels were increased in HNF-3 target genes phosphofructo-2-kinase/fructose-2,6-bisphophatase, insulin growth factor binding protein-1, and hexokinase I of HNF-3α-null mice. Mice lacking one or both HNF-3α alleles also show impaired insulin secretion and glucose intolerance after an intraperitoneal glucose challenge, indicating that pancreatic beta-cell function is also compromised. Our results indicate that HNF-3α plays a critical role in the regulation of glucose homeostasis and in pancreatic islet function.
Transcription repression by
Xenopus
ET and its human ortholog TBX3, a gene involved in ulnar-mammary syndrome
Authors: He Ming-liang , Wen Leng , Campbell Christine E. , Wu Jane Y. , Rao Yi
Published: July 2002

T box
(
Tbx
) genes are a family of developmental regulators with more than 20 members recently identified in invertebrates and vertebrates. Mutations in
Tbx
genes have been found to cause several human diseases. Our understanding of functional mechanisms of
Tbx
products has come mainly from the prototypical
T
/
Brachyury
, which is a transcription activator. We previously discovered
ET
, a
Tbx
gene expressed in
Xenopus
embryos. We report here that
ET
is an ortholog of the human
Tbx3
and that
ET
is a repressor of basal and activated transcription. Functional dissection of the ET protein reveals a novel transcription-repression domain highly conserved among ET, human TBX3, and TBX2. These results reveal a new transcription repressor domain, show the existence of a subfamily of transcription repressors in the
Tbx
superfamily, and provide a basis for understanding etiology of diseases caused by Tbx3 mutations.
Long-term expression of human coagulation factor VIII and correction of hemophilia A after
in vivo
retroviral gene transfer in factor VIII-deficient mice
Authors: VandenDriessche Thierry , Vanslembrouck Veerle , Goovaerts Inge , Zwinnen Hans , Vanderhaeghen Marie-Line , Collen Desire , Chuah Marinee K. L.
Published: July 2002

Hemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and predisposes to spontaneous bleeding that can be life-threatening or lead to chronic disabilities. It is well suited for gene therapy because a moderate increase in plasma FVIII concentration has therapeutic effects. Improved retroviral vectors expressing high levels of human FVIII were pseudotyped with the vesicular stomatitis virus G glycoprotein, were concentrated to high-titers (10
9
–10
10
colony-forming units/ml), and were injected intravenously into newborn, FVIII-deficient mice. High-levels (≥200 milliunits/ml) of functional human FVIII production could be detected in 6 of the 13 animals, 4 of which expressed physiologic or higher levels (500–12,500 milliunits/ml). Five of the six expressers produced FVIII and survived an otherwise lethal tail-clipping, demonstrating phenotypic correction of the bleeding disorder. FVIII expression was sustained for >14 months. Gene transfer occurred into liver, spleen, and lungs with predominant
FVIII
mRNA expression in the liver. Six of the seven animals with transient or no detectable human FVIII developed FVIII inhibitors (7–350 Bethesda units/ml). These findings indicate that a genetic disease can be corrected by
in vivo
gene therapy using retroviral vectors.
Site-directed disulfide bonding reveals an interaction site between energy-coupling protein TonB and BtuB, the outer membrane cobalamin transporter
Authors: Cadieux Nathalie , Kadner Robert J.
Published: July 2002

Transport of vitamin B12across the outer membrane ofEscherichia coli, like that of iron-siderophore complexes, is an active transport process requiring a specific outer membrane transporter BtuB, the proton motive force, and the trans-periplasmic energy coupling protein TonB. Interaction between TonB and two of the TonB-dependent siderophore transporters has been detected previously by formaldehyde crosslinking. Here, site-directed disulfide crosslinking demonstrates contact between a conserved region of BtuB, called the TonB-box, and a portion of TonB, previously implicated as the site of suppressors of TonB-box mutations. The specific pattern of disulfide bonding to alternating residues in the TonB-box allowed deduction of the conformation and parallel orientation of the contact region between these two protein segments. Crosslinking at several positions was increased when BtuB was loaded with substrate, and the crosslinking pattern was altered by the presence of substitutions in BtuB that cause a TonB-uncoupled phenotype. This crosslinking process thus reflects protein interactions that are involved in coupling to active transport.
Paralogous mouse
Hox
genes,
Hoxa9, Hoxb9,
and
Hoxd9,
function together to control development of the mammary gland in response to pregnancy
Authors: Chen Feng , Capecchi Mario R.
Published: July 2002

Although the role of
Hox
genes in patterning the mammalian body plan has been studied extensively during embryonic and fetal development, relatively little is known concerning
Hox
gene function in adult animals. Analysis of mice with mutant
Hoxa9, Hoxb9,
and
Hoxd9
genes shows that these paralogous genes are required for mediating the expansion and/or differentiation of the mammary epithelium ductal system in response to pregnancy. Mothers with these three mutant genes cannot raise their own pups, but the pups can be rescued by fostering by wild-type mothers. Histologically, the mammary glands of the mutant mothers seem normal before pregnancy but do not develop properly in response to pregnancy and parturition.
Hoxa9, Hoxb9,
and
Hoxd9
are expressed normally in adult mammary glands, suggesting a direct role for these genes in the development of mammary tissue after pregnancy. Because loss-of-function mutations in these
Hox
genes cause hypoplasia of the mammary gland after pregnancy, it may be productive to look for misexpression of these genes in mammary carcinomas.
The use of site-directed fluorophore labeling and donor–donor energy migration to investigate solution structure and dynamics in proteins
Authors: Bergström Fredrik , Hägglöf Peter , Karolin Jan , Ny Tor , Johansson Lennart B.-Å.
Published: July 2002

The use of molecular genetics for introducing fluorescent molecules enables the use of donor–donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor–donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the
C
α
-atoms of the positions V106C–H185C, H185C–M266C, and M266C–V106C were 60.9, 30.8, and 55.1 Å, respectively. These are in good agreement with the distances of 54 ± 4, 38 ± 3, and 55 ± 3 Å, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the
C
α
-atoms physically cannot coincide, there is a reasonable agreement between the methods.
RNA-controlled polymorphism in the
in vivo
assembly of 180-subunit and 120-subunit virions from a single capsid protein
Authors: Krol Michael A. , Olson Norman H. , Tate John , Johnson John E. , Baker Timothy S. , Ahlquist Paul
Published: July 2002

Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein–protein interactions. The results presented here reveal a striking
in vivo
switch in CP assembly. Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble
in vivo
two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid. This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP–CP interactions. Thus, the CP of BMV, and perhaps other viruses, can encode CP–CP interactions that are not apparent from mature virions and may function in assembly or disassembly. Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate. The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures. The
in vivo
switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids. RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways.
Identification of transforming growth factor-β- regulated genes in
Caenorhabditis elegans
by differential hybridization of arrayed cDNAs
Authors: Mochii Makoto , Yoshida Satoru , Morita Kiyokazu , Kohara Yuji , Ueno Naoto
Published: July 2002

Members of the transforming growth factor-β family play critical roles in body patterning, in both vertebrates and invertebrates. One transforming growth factor-β-related gene,
dbl-1,
has been shown to regulate body length and male ray patterning in
Caenorhabditis elegans
. We screened arrayed cDNAs to identify downstream target genes for the DBL-1 signaling by using differential hybridization.
C. elegans
cDNAs representing 7,584 independent genes were arrayed on a nylon membrane at high density and hybridized with
33
P-labeled DNA probes synthesized from the mRNAs of wild-type,
dbl-1
,
sma-2,
and
lon-2
worms. Signals for all the spots representing hybridized DNA were quantified and compared among strains. The screening identified 22 and 2 clones, which were positively and negatively regulated, respectively, by the DBL-1 signal. Northern hybridization confirmed the expression profiles of most of the clones, indicating good reliability of the differential hybridization using arrayed cDNAs.
In situ
hybridization analysis revealed the spatial and temporal expression patterns of each clone and showed that at least four genes, including the gene for the type I receptor for DBL-1,
sma-6
, were transcriptionally regulated by the DBL-1 signal.
Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for pRb function are defective only in global repair
Authors: Therrien Jean-Philippe , Drouin Régen , Baril Caroline , Drobetsky Elliot A.
Published: July 2002

After exposure to DNA-damaging agents, the p53 tumor suppressor protects against neoplastic transformation by inducing growth arrest and apoptosis. A series of investigations has also demonstrated that, in UV-exposed cells, p53 regulates the removal of DNA photoproducts from the genome overall (global nucleotide excision repair), but does not participate in an overlapping pathway that removes damage specifically from the transcribed strand of active genes (transcription-coupled nucleotide excision repair). Here, the highly sensitive ligation-mediated PCR was employed to quantify, at nucleotide resolution, the repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in genetically p53-deficient Li–Fraumeni skin fibroblasts, as well as in human lung fibroblasts expressing the human papillomavirus (HPV) E6 oncoprotein that functionally inactivates p53. Lung fibroblasts expressing the HPV E7 gene product, which similarly inactivates the retinoblastoma tumor-suppressor protein (pRb), were also investigated. pRb acts downstream of p53 to mediate G
1
arrest, but has no demonstrated role in DNA repair. Relative to normal cells, HPV E6-expressing lung fibroblasts and Li–Fraumeni skin fibroblasts each manifested defective CPD repair along both the transcribed and nontranscribed strands of the p53 and/or c-
jun
loci. HPV E7-expressing lung fibroblasts also exhibited reduced CPD removal, but only along the nontranscribed strand. Our results provide striking evidence that transcription-coupled repair, in addition to global repair, are p53-dependent in UV-exposed human fibroblasts. Moreover, the observed DNA-repair defect in HPV E7-expressing cells reveals a function for this oncoprotein in HPV-mediated carcinogenesis, and may suggest a role for pRb in global nucleotide excision repair.
Ephrin-dependent growth and pruning of hippocampal axons
Authors: Gao Pan-Pan , Yue Yong , Cerretti Douglas Pat , Dreyfus Cheryl , Zhou Renping
Published: July 2002

Neuronal connections are arranged topographically such that the spatial organization of neurons is preserved by their termini in the targets. During the development of topographic projections, axons initially explore areas much wider than the final targets, and mistargeted axons are pruned later. The molecules regulating these processes are not known. We report here that the ligands of the Eph family tyrosine kinase receptors may regulate both the initial outgrowth and the subsequent pruning of axons. In the presence of ephrins, the outgrowth and branching of the receptor-positive hippocampal axons are enhanced. However, these axons are induced later to degenerate. These observations suggest that the ephrins and their receptors may regulate topographic map formation by stimulating axonal arborization and by pruning mistargeted axons.
Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease
Authors: Gooptu Bibek , Hazes Bart , Chang Wun-Shaing W. , Dafforn Timothy R. , Carrell Robin W. , Read Randy J. , Lomas David A.
Published: July 2002

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.
Putting ion channels to work: Mechanoelectrical transduction, adaptation, and amplification by hair cells
Authors: Hudspeth A. J. , Choe Y. , Mehta A. D. , Martin P.
Published: July 2002

As in other excitable cells, the ion channels of sensory receptors
produce electrical signals that constitute the cellular response to
stimulation. In photoreceptors, olfactory neurons, and some gustatory
receptors, these channels essentially report the results of antecedent
events in a cascade of chemical reactions. The mechanoelectrical
transduction channels of hair cells, by contrast, are coupled directly
to the stimulus. As a consequence, the mechanical properties of these
channels shape our hearing process from the outset of transduction.
Channel gating introduces nonlinearities prominent enough to be
measured and even heard. Channels provide a feedback signal that
controls the transducer's adaptation to large stimuli. Finally,
transduction channels participate in an amplificatory process that
sensitizes and sharpens hearing.
Selectin receptor–ligand bonds: Formation limited by shear rate and dissociation governed by the Bell model
Authors: Chen Shuqi , Springer Timothy A.
Published: July 2002

We have studied the principles that govern the formation and dissociation of an adhesive bond between a cell moving in shear flow and a substrate and tested different theories of how force affects bond dissociation. Viscosity relates the kinematics of fluid movement (shear rate, units of time−1) to shear stress (units of force/area, the product of shear rate and viscosity). At different medium viscosities, the formation of receptor–ligand bonds between a cell in the flowstream and P-selectin on the vessel wall showed a similar efficiency as a function of shear rate but not of shear stress. Therefore, bond formation was a function of shear rate and hence of the kinematics of receptor and ligand movement. By contrast, the kinetics of bond dissociation was a function of shear stress and hence of force on the bond. The different requirements for bond formation and dissociation allowed dissociation kinetics to be measured at higher forces on the bond by increasing medium viscosity. Data over an extended range of forces on the bond therefore could be collected that enabled five different proposed equations, relating force to bond dissociation, to be compared for fit to experimental data. The relationship proposed by Bell [Bell, G. I. (1978)Science200, 618–627] fit the data significantly the best and also predicted an off-rate in the absence of force that best matched an independent measurement [Mehta, P., Cummings, R. D. & McEver, R. P. (1998)J. Biol. Chem.273, 32506–32513].
The Process of Commercialisation of Urban Housing in China
Authors: Ping Wang Ya , Murie Alan
Published: July 2002

The role of the state in housing has been the subject of controversial debate recently in China. More and more decision-makers consider that the supply of housing should be left to market forces of demand and supply. Various new policies have been introduced from as early as 1979, designed to commercialise and reform the public-sector-dominated housing system. This paper provides a review of housing reforms and a systematic account of the key features of the commercialisation process. It focuses principally on the attempts to privatise public-sector housing in urban areas in the context of the major characteristics and problems of the urban housing system, the development of reform policies and legislation and current reform practice.
Parenting in Indonesia: Inter- and intracultural differences in mothers’ interactions with their young children
Authors: Zevalkink Jolien , Riksen-Walraven J. Marianne
Published: July 2002

The interactions between 76 lower-class Indonesian mothers and their young children were observed both at home and in a structured play setting. Maternal interactive behaviour in the play session was compared with that of Japanese, Dutch, and Surinam-Dutch mothers observed in a similar setting. The interactive behaviour of Indonesian mothers was found to be more similar to that of Dutch and Surinam-Dutch mothers than to that of Japanese mothers. Mothers’ supportive behaviour in the structured play session was significantly related to their sensitivity at home. Maternal interactive behaviour in the two settings was also significantly but differentially related to characteristics of the immediate and socioeconomic context. The results suggest that socioeconomic factors have a stronger impact on the quality of parenting than cultural factors and that the observation of mother-child interactions in different settings may tap different aspects of parenting.
A Novel Class of RanGTP Binding Proteins
Authors: Görlich Dirk , Dabrowski Marylena , Bischoff F. Ralf , Kutay Ulrike , Bork Peer , Hartmann Enno , Prehn Siegfried , Izaurralde Elisa
Published: July 2002

The importin-α/β complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of ∼20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-β. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-β, it prevents the activation of Ran's GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-β, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.
Thrombin Stimulates Glucose Transport in Human Platelets via the Translocation of the Glucose Transporter GLUT-3 from α-Granules to the Cell Surface
Authors: Heijnen Harry F.G. , Oorschot Viola , Sixma Jan J. , Slot Jan W. , James David E.
Published: July 2002

Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most α-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the α-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant α-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of α-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in α-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (∼15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.
Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant
Authors: Luo Wen-jie , Chang Amy
Published: July 2002

A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.
A Dual-Specificity Phosphatase Cdc25B Is an Unstable Protein and Triggers p34cdc2/Cyclin B Activation in Hamster BHK21 Cells Arrested with Hydroxyurea
Authors: Nishijima Hitoshi , Nishitani Hideo , Seki Takashi , Nishimoto Takeharu
Published: July 2002

By incubating at 30°C in the presence of an energy source, p34cdc2/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40°C, the nonpermissive temperature (Nishimoto, T., E. Eilen, and C. Basilico. 1978. Cell. 15:475–483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34cdc2/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34cdc2/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40°C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34cdc2/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34cdc2/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34cdc2/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34cdc2/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34cdc2/cyclin B, which initiates mitosis through the activation of Cdc25C.
Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris
Authors: Waterham Hans R. , Russell Kimberly A. , Vries Yne de , Cregg James M.
Published: July 2002

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX–HA). Coexpression of AOX–HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX–HA present in octameric active AOX, suggesting that newly synthesized AOX–HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.
Calpain Regulates Actin Remodeling during Cell Spreading
Authors: Potter David A. , Tirnauer Jennifer S. , Janssen Richard , Croall Dorothy E. , Hughes Christina N. , Fiacco Kerry A. , Mier James W. , Maki Masatoshi , Herman Ira M.
Published: July 2002

Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3– derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.
Analysis of Cd44-Containing Lipid Rafts
Authors: Oliferenko Snezhana , Paiha Karin , Harder Thomas , Gerke Volker , Schwärzler Christoph , Schwarz Heinz , Beug Hartmut , Günthert Ursula , Huber Lukas A.
Published: July 2002

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II–p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II–lipid raft complexes were stabilized by addition of GTPγS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.