bluuebunny/crossref_metadata_2025 huggingface dataset:
The early Upper Paleolithic human skeleton from the Abrigo do Lagar Velho (Portugal) and modern human emergence in Iberia
Authors: Duarte Cidália , Maurício João , Pettitt Paul B. , Souto Pedro , Trinkaus Erik , van der Plicht Hans , Zilhão João
Published: July 2002

The discovery of an early Upper Paleolithic human burial at the Abrigo do Lagar Velho, Portugal, has provided evidence of early modern humans from southern Iberia. The remains, the largely complete skeleton of a ≈4-year-old child buried with pierced shell and red ochre, is dated to
ca
. 24,500 years B.P. The cranium, mandible, dentition, and postcrania present a mosaic of European early modern human and Neandertal features. The temporal bone has an intermediate-sized juxtamastoid eminence. The mandibular mentum osseum and the dental size and proportions, supported by mandibular ramal features, radial tuberosity orientation, and diaphyseal curvature, as well as the pubic proportions align the skeleton with early modern humans. Body proportions, reflected in femorotibial lengths and diaphyseal robusticity plus tibial condylar displacement, as well as mandibular symphyseal retreat and thoracohumeral muscle insertions, align the skeleton with the Neandertals. This morphological mosaic indicates admixture between regional Neandertals and early modern humans dispersing into southern Iberia. It establishes the complexities of the Late Pleistocene emergence of modern humans and refutes strict replacement models of modern human origins.
Endogenous presenilin 1 redistributes to the surface of lamellipodia upon adhesion of Jurkat cells to a collagen matrix
Authors: Schwarzman Alexander L. , Singh Nandita , Tsiper Maria , Gregori Luisa , Dranovsky Alex , Vitek Michael P. , Glabe Charles G. , St. George-Hyslop Peter H. , Goldgaber Dmitry
Published: July 2002

Most familial early-onset Alzheimer’s disease cases are caused by mutations in the presenilin 1 (PS1) gene. Subcellular localization of the endogenous PS1 is essential for understanding its function, interactions with proteins, and role in Alzheimer’s disease. Although numerous studies revealed predominant localization of PS1 to endoplasmic reticulum and Golgi, there are conflicting reports on the localization of PS1 to the cell surface. We found that endogenous PS1 is highly expressed in T lymphocytes (Jurkat cells). Using a variety of methods, we present evidence that endogenous PS1 is localized to the cell surface in addition to intracellular membrane compartments. Moreover, PS1 appeared in high levels on the surface of lamellipodia upon adhesion of the cells to a collagen matrix. The redistribution of PS1 in adhered cells was strikingly similar to that of the well characterized adhesion protein CD44. Cell surface PS1 formed complexesin vivowith actin-binding protein filamin (ABP-280), which is known to form bridges between cell surface receptors and cytoskeleton and mediate cell adhesion and cell motility. Taken together, our results suggest a role of PS1 in cell adhesion and/or cell–matrix interaction.
Genetic elimination of known pheromones reveals the fundamental chemical bases of mating and isolation in
Drosophila
Authors: Savarit Fabrice , Sureau Gilles , Cobb Matthew , Ferveur Jean-François
Published: July 2002

Overexpression of the
UAS-tra
transgene in
Drosophila melanogaster
females led to the complete elimination of their cuticular pheromones. According to current models of
Drosophila
behavior, these flies should induce no courtship. In fact, they are still attractive to conspecific males. Three classes of stimuli are shown to induce courtship, with different effects on male behavior: (
i
) known pheromones produced by control females, (
ii
) stimuli produced by living control and transgenic flies, and (
iii
) as-yet-undetermined pheromones present on both control and transgenic flies. Only the latter class of pheromones are required for mating. They appear to represent a layer of ancestral attractive substances present in
D. melanogaster
and its sibling species; known cuticular pheromones modulate this attractivity positively or negatively. The absence of inhibitory pheromones leads to high levels of interspecific mating, suggesting an important role for these cuticular hydrocarbons in isolation between species.
Heritability of body mass, a sexually selected trait, in an arctiid moth (
Utetheisa ornatrix
)
Authors: Iyengar Vikram K. , Eisner Thomas
Published: July 2002

In the moth
Utetheisa ornatrix
(Lepidoptera: Arctiidae), females mate preferentially with larger males. Large body mass is advantageous to both sexes: large males sire more young than small males, and large females have higher fecundity than small females. Here we report that body mass is heritable in both sexes, indicating that by choosing larger males females obtain genetic benefits for their offspring. Choosy females also receive extra nutrient and defensive alkaloid by way of their partner’s spermatophores, but these gifts do not affect the heritability of body mass. These results indicate that by exercising mate choice female
Utetheisa
receive both direct phenotypic and indirect genetic benefits.
A silencer element identified in
Drosophila
is required for imprinting of
H19
reporter transgenes in mice
Authors: Brenton J. D. , Drewell R. A. , Viville S. , Hilton K. J. , Barton S. C. , Ainscough J. F-X. , Surani M. A.
Published: July 2002

The
H19
gene is subject to genomic imprinting because it is methylated and repressed after paternal inheritance and is unmethylated and expressed after maternal inheritance. We recently identified a 1.1-kb control element in the upstream region of the
H19
gene that functions as a cis
-
acting silencer element in
Drosophila
. Here we investigate the function of this element in mice. We demonstrate that both
H19–lacZ
and
H19–PLAP
reporter transgenes can undergo imprinting with repression and hypermethylation after paternal transmission at many integration sites. However, transgenes that were deleted for the 1.1-kb silencer element showed loss of paternal repression, but they did not show marked changes in the paternal methylation of the remaining upstream region. This study demonstrates that the 1.1-kb control element identified in
Drosophila
is required to silence paternally transmitted
H19
minitransgenes in mice.
Myosin-dependent contractile activity of the actin cytoskeleton modulates the spatial organization of cell–cell contacts in cultured epitheliocytes
Authors: Krendel M. , Gloushankova N. A. , Bonder E. M. , Feder H. H. , Vasiliev J. M. , Gelfand I. M.
Published: July 2002

The spatial organization of cell–cell adherens junctions is distinct in cultured cells from two different tissue types, specifically, epitheliocytes and fibroblasts. In epitheliocytes, contacts are localized tangentially, along contacting cell edges and in association with circumferential actin bundles. Contacts between fibroblasts are radially oriented; that is, they are perpendicular to the overlapping edges of the cells and are associated with straight bundles of actin filaments. In the present study, we establish that the spatial organization of cell–cell contacts in the epithelial cell line IAR-2 can be converted from the typical tangential pattern to the radial pattern observed in fibroblasts. This transition can be induced by treatment with two agents, phorbol 12-myristate 13-acetate and nocodazole, which have different modes of action. Inhibition of myosin contractility reverses tangential-to-radial conversion of cell–cell contacts. These data suggest that formation of radially aligned contacts depends on modulation of contractility within the actin cytoskeleton through the myosin motor protein. The results open the possibility that modulation of the spatial organization of cell–cell contacts may play important roles in regulating organization and physiological functions of epithelial tissues.
Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin
Authors: Grunwald Maria E. , Zhong Haining , Lai Jun , Yau King-Wai
Published: July 2002

The action of calmodulin (CaM) on target proteins is
important for a variety of cellular functions. We demonstrate here,
however, that the presence of a CaM-binding site on a protein does not
necessarily imply a functional effect. The α-subunit of the
cGMP-gated cation channel of human retinal cones has a CaM-binding site
on its cytoplasmic N-terminal region, but the homomeric channel that it
forms is not functionally modulated by CaM. Mutational analysis based
on comparison to the highly homologous olfactory cyclic
nucleotide-gated channel α-subunit, which does form a CaM-modulated
channel, indicates that residues downstream of the CaM-binding domain
on these channels are also important for CaM to have an effect. These
findings suggest that a CaM-binding site and complementary structural
features in a protein probably evolve independently, and an effect
caused by CaM occurs only in the presence of both elements. More
generally, the same may be true for other recognized binding sites on
proteins for modulators or activators, so that a demonstrated physical
interaction does not necessarily imply functional consequence.
The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules
Authors: Chan Jordi , Jensen Cynthia G. , Jensen Lawrence C. W. , Bush Max , Lloyd Clive W.
Published: July 2002

In plants, cortical microtubules (MTs) occur in characteristically parallel groups maintained up to one microtubule diameter apart by fine filamentous cross-bridges. However, none of the plant microtubule-associated proteins (MAPs) so far purified accounts for the observed separation between MTs in cells. We previously isolated from carrot cytoskeletons a MAP fraction including 120- and 65-kDa MAPs and have now separated the 65-kDa carrot MAP by sucrose density centrifugation. MAP65 does not induce tubulin polymerization but induces the formation of bundles of parallel MTs in a nucleotide-insensitive manner. The bundling effect is inhibited by porcine MAP2, but, unlike MAP2, MAP65 is heat-labile. In the electron microscope, MAP65 appears as filamentous cross-bridges, maintaining an intermicrotubule spacing of 25–30 nm. Microdensitometer-computer correlation analysis reveals that the cross-bridges are regularly spaced, showing a regular axial spacing that is compatible with a symmetrical helical superlattice for 13 protofilament MTs. Because MAP65 maintains
in vitro
the inter-MT spacing observed in plants and is shown to decorate cortical MTs, it is proposed that this MAP is important for the organization of the cortical array
in vivo
.
Expression of DFak56, a
Drosophila
homolog of vertebrate focal adhesion kinase, supports a role in cell migration
in
vivo
Authors: Fox George L. , Rebay Ilaria , Hynes Richard O.
Published: July 2002

Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein–protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration.
Interaction of 5-lipoxygenase with cellular proteins
Authors: Provost Patrick , Samuelsson Bengt , Rådmark Olof
Published: July 2002

5-Lipoxygenase (5LO) plays a pivotal role in cellular leukotriene synthesis. To identify proteins interacting with human 5LO, we used a two-hybrid approach to screen a human lung cDNA library. From a total of 1.5 × 10
7
yeast transformants, nine independent clones representing three different proteins were isolated and found to specifically interact with 5LO. Four 1.7- to 1.8-kb clones represented a 16-kDa protein named coactosin-like protein for its significant homology with coactosin, a protein found to be associated with actin in
Dictyostelium discoideum
.
Coactosin-like protein thus may provide a link between 5LO and the cytoskeleton. Two other yeast clones of 1.5 kb encoded transforming growth factor (TGF) type β receptor-I-associated protein 1 partial cDNA. TGF type β receptor-I-associated protein 1 recently has been reported to associate with the activated form of the TGF β receptor I and may be involved in the TGF β-induced up-regulation of 5LO expression and activity observed in HL-60 and Mono Mac 6 cells. Finally, three identical 2.1-kb clones contained the partial cDNA of a human protein with high homology to a hypothetical helicase K12H4.8 from
Caenorhabditis elegans
and consequently was named ΔK12H4.8 homologue. Analysis of the predicted amino acid sequence revealed the presence of a RNase III motif and a double-stranded RNA binding domain, indicative of a protein of nuclear origin. The identification of these 5LO-interacting proteins provides additional approaches to studies of the cellular functions of 5LO.
Transcriptional activation by artificial recruitment in mammalian cells
Authors: Nevado Julián , Gaudreau Luc , Adam Maryse , Ptashne Mark
Published: July 2002

We show that the typical “nonclassical” activator, which comprises a fusion protein bearing a component of the transcriptional machinery fused to a DNA-binding domain, activates transcription in mammalian cells only weakly when tested with an array of promoters. However, as found in analogous “artificial recruitment” experiments performed in yeast, these activators work synergistically with “classical” activators. The effect of the classical activator in such experiments requires that it be tethered to DNA, a requirement that cannot be overcome by expression of that classical activator at high levels. The effect of the one nonclassical activator that does elicit significant levels of transcription when working alone (i.e., that bearing TATA box-binding protein) is strongly influenced by promoter architecture. The results, consistent with those of analogous experiments in yeast [see the accompanying paper: Gaudreau, L., Keaveney, M., Nevado, J., Zaman, Z., Bryant, G. O., Struhl, K. & Ptashne, M. (1999)
Proc. Natl. Acad. Sci. USA
96, 2668–2673], suggest that classical activators, presumably by virtue of their abilities to interact with multiple targets, have a functional flexibility that nonclassical activators lack.
In vitrocomparison of the antigen-binding and stability properties of the various molecular forms of IgA antibodies assembled and produced in CHO cells
Authors: Berdoz José , Blanc Corinne Tallichet , Reinhardt Monique , Kraehenbuhl Jean-Pierre , Corthésy Blaise
Published: July 2002

The hallmark of a mucosal immune response is the production of antigen-specific secretory IgA (S-IgA) antibodies in external secretions. S-IgA consists of ten polypeptides produced in two different cell lineages. The heavy and light chains in plasma cells assemble into IgA, which on association with J chain become polymerized, whereas secretory component (SC) is added during transport across the epithelium. Recombinant chimeric mouse–human monomeric, dimeric, and S-IgA antibodies have been produced in a single CHO cell sequentially transfected with expression vectors carrying three independent selective markers for chimeric heavy and light chains, human J chain, and human SC, respectively. Biochemical characterization of the various molecular forms indicates that the assembly of the various polypeptides resulted in species of the expected size and covalence. All chimeric IgA antibodies retained the antigen-binding capacity of the parent mouse IgA antibody. The resistance of S-IgA to protease-rich intestinal washes was enhanced when compared with dimeric IgA lacking associated SC. Up to 20 μg of recombinant S-IgA per 1 × 106cells were recovered in 24 h with the best producing clones. We conclude that CHO cells programmedde novowith four different genetic elements can assemble functional chimeric S-IgA.
Induction of utrophin gene expression by heregulin in skeletal muscle cells: Role of the N-box motif and GA binding protein
Authors: Gramolini Anthony O. , Angus Lindsay M. , Schaeffer Laurent , Burton Edward A. , Tinsley Jonathon M. , Davies Kay E. , Changeux Jean-Pierre , Jasmin Bernard J.
Published: July 2002

The modulation of utrophin gene expression in muscle by the nerve-derived factor agrin plausibly involves the trophic factor ARIA/heregulin. Here we show that heregulin treatment of mouse and human cultured myotubes caused a ≈2.5-fold increase in utrophin mRNA levels. Transient transfection experiments with utrophin promoter-reporter gene constructs showed that this increase resulted from an enhanced transcription of the utrophin gene. In the case of the nicotinic acetylcholine receptor δ and ɛ subunit genes, heregulin was previously reported to stimulate transcription via a conserved promoter element, the N-box, which binds the multimeric Ets-related transcription factor GA binding protein (GABP). Accordingly, site-directed mutagenesis of a single N-box motif in the utrophin gene promoter abolished the transcriptional response to heregulin. In addition, overexpression of heregulin, or of the two GABP subunits in cultured myotubes, caused an N-box-dependent increase of the utrophin promoter activity.
In vivo
, direct gene transfer into muscle confirmed that heregulin regulates utrophin gene expression. Finally, electrophoretic mobility shift assays and supershift experiments performed with muscle extracts revealed that the N-box of the utrophin promoter binds GABP. These findings suggest that the subsynaptic activation of transcription by heregulin via the N-box motif and GABP are conserved among genes expressed at the neuromuscular junction. Because utrophin can functionally compensate for the lack of dystrophin, the elucidation of the molecular mechanisms regulating utrophin gene transcription may ultimately lead to therapies based on utrophin expression throughout the muscle fibers of Duchenne muscular dystrophy patients.
Association of a missense change in the D2 dopamine receptor with myoclonus dystonia
Authors: Klein Christine , Brin Mitchell F. , Kramer Patricia , Sena-Esteves Miguel , de Leon Deborah , Doheny Dana , Bressman Susan , Fahn Stanley , Breakefield Xandra O. , Ozelius Laurie J.
Published: July 2002

Hereditary autosomal dominant myoclonus dystonia (MD) is a movement disorder characterized by involuntary lightning jerks and dystonic movements and postures alleviated by alcohol. Although various large families with MD have been described, no positive linkage has been found to a chromosomal location. We report a family with eight members with MD. Linkage analysis identified a 23-centimorgan region on chromosome 11q23 that cosegregates with the disease state (maximum multipoint logarithm of odds score = 2.96 at D11S897). This region contains an excellent candidate gene for involvement in the etiology of MD, the D2 dopamine receptor (
DRD2
) gene. Neurotransmission mediated by
DRD2
is known to have a key role in the control of movement and also has been implicated in reward and reinforcement mechanisms and psychiatric disorders. Sequencing of the coding region of
DRD2
indicated that all affected and obligate carriers were heterozygous for a Val154Ile change in exon 3 of the protein, which is highly conserved across species. This change was found neither in other unaffected members of the pedigree nor in 250 control chromosomes. Our finding provides evidence for the involvement of
DRD2
in a disorder of the central nervous system and should lead to further insight into the function of the dopaminergic system in dystonia and other movement and mood disorders.
Diversity, functionality, and stability of the T cell repertoire derived
in vivo
from a single human T cell precursor
Authors: Bousso Philippe , Wahn Volker , Douagi Iyadh , Horneff Gerd , Pannetier Christophe , Le Deist Françoise , Zepp Fred , Niehues Tim , Kourilsky Philippe , Fischer Alain , de Saint Basile Geneviève
Published: July 2002

In this report, we have analyzed the human T cell repertoire derived
in vivo
from a single T cell precursor. A unique case of X-linked severe combined immunodeficiency in which a reverse mutation occurred in an early T cell precursor was analyzed to this end. It was determined that at least 1,000 T cell clones with unique T cell receptor-β sequences were generated from this precursor. This diversity seems to be stable over time and provides protection from infections
in vivo
. A similar estimation was obtained in an
in vitro
murine model of T cell generation from a single cell precursor. Overall, our results document the large diversity potential of T cell precursors and provide a rationale for gene therapy of the block of T cell development.
Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target
Authors: Todone Flavia , Weinzierl Robert O. J. , Brick Peter , Onesti Silvia
Published: July 2002

Eukaryotic nuclei contain three different types of RNA polymerases (RNAPs), each consisting of 12–18 different subunits. The evolutionarily highly conserved RNAP subunit RPB5 is shared by all three enzymes and therefore represents a key structural/functional component of all eukaryotic RNAPs. Here we present the crystal structure of the RPB5 subunit from
Saccharomyces cerevisiae
. The bipartite structure includes a eukaryote-specific N-terminal domain and a C-terminal domain resembling the archaeal RNAP subunit H. RPB5 has been implicated in direct protein-protein contacts with transcription factor IIB, one of the components of the RNAP
II
basal transcriptional machinery, and gene-specific activator proteins, such as the hepatitis B virus transactivator protein X. The experimentally mapped regions of RPB5 involved in these interactions correspond to distinct and surface-exposed α-helical structures.
Circadian rise in maternal glucocorticoid prevents pulmonary dysplasia in fetal mice with adrenal insufficiency
Authors: Venihaki Maria , Carrigan Allison , Dikkes Pieter , Majzoub Joseph A.
Published: July 2002

The hypothalamic-pituitary-adrenal (HPA) axis, including hypothalamic corticotropin-releasing hormone (CRH) and pituitary corticotropin, is one of the first endocrine systems to develop during fetal life, probably because glucocorticoid secretion is necessary for the maturation of many essential fetal organs. Consistent with this, pregnant mice with an inactivating mutation in the
Crh
gene deliver CRH-deficient offspring that die at birth with dysplastic lungs, which can be prevented by prenatal maternal glucocorticoid treatment. But children lacking the ability to synthesize cortisol (because of various genetic defects in adrenal gland development or steroidogenesis) are not born with respiratory insufficiency or abnormal lung development, suggesting that the transfer of maternal glucocorticoid across the placenta might promote fetal organ maturation in the absence of fetal glucocorticoid production. We used pregnant mice with a normal HPA axis carrying fetuses with CRH deficiency to characterize the relative contributions of the fetal and maternal adrenal to the activity of the fetal HPA axis, and related these findings to fetal lung development. We found that in the presence of fetal adrenal insufficiency, normal fetal lung development is maintained by the transfer of maternal glucocorticoid to the fetus, specifically during the circadian peak in maternal glucocorticoid secretion.
Action of the
Caenorhabditis elegans
GATA factor END-1 in
Xenopus
suggests that similar mechanisms initiate endoderm development in ecdysozoa and vertebrates
Authors: Shoichet Sarah A. , Malik Talat H. , Rothman Joel H. , Shivdasani Ramesh A.
Published: July 2002

In ecdysozoan protostomes, including arthropods and nematodes, transcription factors of the GATA family specify the endoderm:
Drosophila
dGATAb (ABF/Serpent) and
Caenorhabditis elegans
END-1 play important roles in generating this primary germ layer.
end-1
is the earliest expressed endoderm-specific gene known in
C. elegans
and appears to initiate the program of gene expression required for endoderm differentiation, including a cascade of GATA factors required for development and maintenance of the intestine. Among vertebrate GATA proteins, the GATA-4/5/6 subfamily regulates aspects of late endoderm development, but a role for GATA factors in establishing the endoderm is unknown. We show here that END-1 binds to the canonical target DNA sequence WGATAR with specificity similar to that of vertebrate GATA-1 and GATA-4, and that it functions as a transcriptional activator. We exploited this activity of END-1 to demonstrate that establishment of the vertebrate endoderm, like that of invertebrate species, also appears to involve GATA transcriptional activity. Like the known vertebrate endoderm regulators Mixer and Sox17, END-1 is a potent activator of endoderm differentiation in isolated
Xenopus
ectoderm. Moreover, a dominant inhibitory GATA-binding fusion protein abrogates endoderm differentiation in intact embryos. By examining these effects in conjunction with those of Mixer- and Sox17β-activating and dominant inhibitory constructs, we further establish the likely relationships between GATA activity and these regulators in early development of the vertebrate endoderm. These results suggest that GATA factors may function sequentially to regulate endoderm differentiation in both protostomes and deuterostomes.
Ecological character displacement inPlethodon: Biomechanical differences found from a geometric morphometric study
Authors: Adams Dean C. , Rohlf F. James
Published: July 2002

Ecological character displacement describes a pattern where morphological differences between sympatric species are enhanced through interspecific competition. Although widely considered a pervasive force in evolutionary ecology, few clear-cut examples have been documented. Here we report a case of ecological character displacement between two salamander species,Plethodon cinereusandPlethodon hoffmani.Morphology was quantified by using linear measurements and landmark-based geometric morphometric methods for specimens from allopatric and sympatric populations from two geographic transects in south-central Pennsylvania, and stomach contents were assayed to quantify food resource use. Morphological variation was also assessed in 13 additional allopatric populations. In both transects, we found significant morphological differentiation between sympatric populations that was associated with a reduction in prey consumption in sympatry and a segregation of prey according to prey size. No trophic morphological or resource use differences were found between allopatric populations, and comparisons of sympatric populations with randomly paired allopatric populations revealed that the observed sympatric morphological differentiation was greater than expected by chance. The major trophic anatomical differences between sympatric populations relates to functional and biomechanical differences in jaw closure: sympatricP. hoffmanihave a faster closing jaw, whereas sympatricP. cinereushave a slower, stronger jaw. Because salamanders immobilize prey of different sizes in different ways, and because the observed sympatric biomechanical differences in jaw closure are associated with the differences in prey consumption, the observed character displacement has a functional ecological correlate, and we can link changes in form with changes in function in this apparent example of character displacement.
Reason for Medical Hospitalization Among Adult Alcohol and Drug Abusers
Authors: Weintraub Eric , Dixon Lisa , Delahanty Janine , Schwartz Robert , Johnson Jeannette , Cohen Arthur , Klecz Mary
Published: July 2002

This study attempts to identify the associations between types of substance use and particular medical problems as causes of acute hospital admission on an inpatient substance abuse consultation service. Records of all consultations performed from 1994 to 1998 were analyzed. A total of 4,526 complete records were available. Cocaine (p < .01), heroin (p< .001), and injection drug (p< .001) users were more likely to be admitted to the hospital for infection. Both alcohol (p< .001) and marijuana (p < .001) users were more likely to be hospitalized for trauma related injuries. Individuals that used alcohol were also more likely to be admitted for the treatment of gastrointestinal disorders (p < .001). Clinicians who treat patients with these diagnoses should have a high index of suspicion of co‐morbid substance use disorders. Effective treatment of substance use disorders can lead to a decrease in medical morbidity, improved outcomes for individual patients, and decreased costs for the health care system.
Phenomenology of Gasoline Intoxication and Withdrawal Symptoms Among Adolescent in India: A Case Series
Authors: Shah Rakesh , Vankar G.K. , Upadhyaya Himanshu P.
Published: July 2002

Inhalant withdrawal symptoms have previously been described but not well documented. This case‐series describes the socio‐demographic and clinical characteristics of nine children and adolescents with gasoline abuse. Gasoline inhalation was the drug of choice in our patients. The mean age was 13.6 years and all the subjects belonged to low socioeconomic status. Most of the subjects used daily and all subjects reported alcohol abuse in the father. All subjects reported a syndrome of intoxication as described in previous reports. All subjects also reported a withdrawal syndrome including irritability, psychomotor retardation, anhedonia, dry mouth, sleep disturbances, craving, and increased lacrimation.
Ethanol metabolism by macrophages: possible role in organ damage
Authors: Wickramasinghe S. N.
Published: July 2002

Macrophages and hepatocytes oxidize ethanol to acetate in vitro at comparable rates but by different biochemical pathways. Ethanol metabolism by macrophages is largely ADH‐independent and mainly based on cytochrome P450 and on the extracellular release of superoxide anion radicals. There is also evidence that during ethanol metabolism, macrophages release more acetaldehyde extracellularly than hepatocytes; the high concentrations of acetaldehyde around macrophages may damage surrounding tissue cells. Some of this acetaldehyde forms unstable cytotoxic complexes with serum albumin and with erythrocytes. The superoxide anion radicals released by macrophages may not only oxidize ethanol to acetaldehyde but also react with and damage cells in their immediate vicinity. After exposure to ethanol, macrophage‐depleted rodents show markedly reduced levels of cytotoxic acetaldehyde‐albumin complexes in the blood and reduced levels of hydroxyethyl radicals in the bile compared to control animals, indicating that the generation of such potentially pathogenic molecules is, to a large extent, dependent on macrophage activity. Macrophage‐depleted animals also show less early liver damage than control animals. The reduction in ethanol‐induced liver damage in macrophage‐depleted mice and rats may be due to a reduction or elimination of the generation of various Kupffer‐cell‐derived hepatotoxic substances, including acetaldehyde and reactive oxygen radicals, in such animals. These data suggest that ethanol metabolism by tissue macrophages may play an important role in mediating ethanol‐related tissue damage.
Using an interdisciplinary approach to training to develop the quality of communication with adults with profound learning disabilities by care staff
Authors: Dobson Susan , Upadhyaya Shripati , Stanley Brian
Published: July 2002

The study examines the effects of training on care staff's communication to service users with profound and severe learning disabilities. An interdisciplinary therapeutic training programme aimed to improve care staff's cognitions about disability, to alter their communicative practice and to provide them with practical solutions to the communication difficulties of the service users. Participants were each video recorded with a single service user before and during training. The recordings formed the basis of the communication workshops. Both baseline and final recordings were evaluated using criteria based on verbal and non‐verbal interactions. The criteria assessed alterations in the care staff's language use, verbal responses, and interpretation and praise of service users' communication. The use of posture, position, eye contact and gaze monitoring during interactions was also evaluated. The pre‐ and post‐training analysis demonstrated alterations and improvements in care staff's use of verbal communication as well as gaze monitoring and position during interactions. A follow‐up study 6 months later showed many of the post‐training positive changes had been maintained or had continued to develop. It is suggested that the success and implementation of the communication training in the workplace was due to the collaborative therapeutic nature of the training package. The interdisciplinary and integrated training programme had facilitated a willingness to change and led to the development of a positive view of each other's practice. The recommendations from each of the therapist's training programme formed an integral part of the care staff's new care plans for each service user. This contributed to the continuing development the care staff's therapeutic role.
Dynamic Properties of an Inositol 1,4,5-Trisphosphate– and Thapsigargin-insensitive Calcium Pool in Mammalian Cell Lines
Authors: Pizzo Paola , Fasolato Cristina , Pozzan Tullio
Published: July 2002

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate– and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate–sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+–ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+–ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.
The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes
Authors: Wilson Nedra F. , Foglesong Mary J. , Snell William J.
Published: July 2002

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ∼3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt−) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt− gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2–3 × 106 fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt− gametes between the two flagella, the site of the mt− mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt− gametes.
Phospholipase D Stimulates Release of Nascent Secretory Vesicles from the trans-Golgi Network
Authors: Chen Ye-Guang , Siddhanta Anirban , Austin Cary D. , Hammond Scott M. , Sung Tsung-Chang , Frohman Michael A. , Morris Andrew J. , Shields Dennis
Published: July 2002

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.
Suppression of KIF2 in PC12 Cells Alters the Distribution of a Growth Cone Nonsynaptic Membrane Receptor and Inhibits Neurite Extension
Authors: Morfini Gerardo , Quiroga Santiago , Rosa Alberto , Kosik Kenneth , Cáceres Alfredo
Published: July 2002

In the present study, we present evidence about the cellular functions of KIF2, a kinesin-like superfamily member having a unique structure in that its motor domain is localized at the center of the molecule (Noda Y., Y. Sato-Yoshitake, S. Kondo, M. Nangaku, and N. Hirokawa. 1995. J. Cell Biol. 129:157–167.). Using subcellular fractionation techniques, isopicnic sucrose density centrifugation of microsomal fractions from developing rat cerebral cortex, and immunoisolation with KIF2 antibodies, we have now identified a type of nonsynaptic vesicle that associates with KIF2. This type of organelle lacks synaptic vesicle markers (synapsin, synaptophysin), amyloid precursor protein, GAP-43, or N-cadherin. On the other hand, it contains βgc, which is a novel variant of the β subunit of the IGF-1 receptor, which is highly enriched in growth cone membranes. Both βgc and KIF2 are upregulated by NGF in PC12 cells and highly concentrated in growth cones of developing neurons. We have also analyzed the consequences of KIF2 suppression by antisense oligonucleotide treatment on nerve cell morphogenesis and the distribution of synaptic and nonsynaptic vesicle markers. KIF2 suppression results in a dramatic accumulation of βgc within the cell body and in its complete disappearance from growth cones; no alterations in the distribution of synapsin, synaptophysin, GAP-43, or amyloid percursor protein are detected in KIF2-suppressed neurons. Instead, all of them remained highly enriched at nerve terminals. KIF2 suppression also produces a dramatic inhibition of neurite outgrowth; this phenomenon occurs after βgc has disappeared from growth cones. Taken collectively, our results suggest an important role for KIF2 in neurite extension, a phenomenon that may be related with the anterograde transport of a type of nonsynaptic vesicle that contains as one of its components a growth cone membrane receptor for IGF-1, a growth factor implicated in nerve cell development.
Active Site Mutations in Yeast Protein Disulfide Isomerase Cause Dithiothreitol Sensitivity and a Reduced Rate of Protein Folding in the Endoplasmic Reticulum
Authors: Holst Bjørn , Tachibana Christine , Winther Jakob R.
Published: July 2002

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a′, each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a′ domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.
Isolation of Functional Golgi-derived Vesicles with a Possible Role in Retrograde Transport
Authors: Love Harold D. , Lin Chung-Chih , Short Craig S. , Ostermann Joachim
Published: July 2002

Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis-side and exit in transport vesicles budding from the trans-side. Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretory flow must be recycled constantly by retrograde transport in the opposite direction. In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells. We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cisternal membranes. These vesicles appeared to be depleted of secretory cargo. They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus. Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack.
E4orf4, a Novel Adenovirus Death Factor That Induces p53-independent Apoptosis by a Pathway That Is Not Inhibited by zVAD-fmk
Authors: Lavoie Josée N. , Nguyen M. , Marcellus R.C. , Branton P.E. , Shore G.C.
Published: July 2002

In the absence of E1B, the 289-amino acid product of human adenovirus type 5 13S E1A induces p53-independent apoptosis by a mechanism that requires viral E4 gene products (Marcellus, R.C., J.C. Teodoro, T. Wu, D.E. Brough, G. Ketner, G.C. Shore, and P.E. Branton. 1996. J. Virol. 70:6207–6215) and involves a mechanism that includes activation of caspases (Boulakia, C.A., G. Chen, F.W. Ng, J.G. Teodoro, P.E. Branton, D.W. Nicholson, G.G. Poirier, and G.C. Shore. 1996. Oncogene. 12:529–535). Here, we show that one of the E4 products, E4orf4, is highly toxic upon expression in rodent cells regardless of the p53 status, and that this cytotoxicity is significantly overcome by coexpression with either Bcl-2 or Bcl-Xl. Conditional expression of E4orf4 induces a cell death process that is characterized by apoptotic hallmark features, such as externalization of phosphatidylserine, loss of mitochondrial membrane potential, cytoplasmic vacuolation, condensation of chromatin, and internucleosomal DNA degradation. However, the wide-spectrum inhibitor of caspases, tetrapeptide zVAD-fmk, does not affect any of these apoptogenic manifestations, and does not alter the kinetics of E4orf4-induced cell death. Moreover, E4orf4 expression does not result in activation of the downstream effector caspase common to most apoptosis-inducing events, caspase-3 (CPP32). We conclude, therefore, that in the absence of E1A, E4orf4 is sufficient by itself to trigger a p53-independent apoptosis pathway that may operate independently of the known zVAD-inhibitable caspases, and that may involve an as yet uncharacterized mechanism.
The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells
Authors: Chuang Jen-Zen , Sung Ching-Hwa
Published: July 2002

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.