bluuebunny/crossref_metadata_2025 huggingface dataset:
Highly stable expression of a foreign gene from rabies virus vectors.
Authors: Mebatsion T , Schnell M J , Cox J H , Finke S , Conzelmann K K
Published: July 2002

A reverse genetics approach was applied to generate a chimeric nonsegmented negative strand RNA virus, rabies virus (RV) of the Rhabdoviridae family, that expresses a foreign protein. DNA constructs containing the entire open reading frame of the bacterial chloramphenicol acetyltransferase (CAT) gene and an upstream RV cistron border sequence were inserted either into the nontranslated pseudogene region of a full-length cDNA copy of the RV genome or exchanged with the pseudogene region. After intracellular T7 RNA polymerase-driven expression of full-length antigenome RNA transcripts and RV nucleoprotein, phosphoprotein and polymerase from transfected plasmids, RVs transcribing novel monocistronic mRNAs and expressing CAT at high levels, were recovered. The chimeric viruses possessed the growth characteristics of standard RV and were genetically stable upon serial cell culture passages. CAT activity was still observed in cell cultures infected with viruses passaged for more than 25 times. Based on the unprecedented stability of the chimeric RNA genomes, which is most likely due to the structure of the rhabdoviral ribonucleoprotein complex, we predict the successful future use of recombinant rhabdovirus vectors for displaying foreign antigens or delivering therapeutic genes.
Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions.
Authors: Prymakowska-Bosak M , Przewłoka M R , Iwkiewicz J , Egierszdorff S , Kuraś M , Chaubet N , Gigot C , Spiker S , Jerzmanowski A
Published: July 2002

Histone H1, a major structural component of chromatin fiber, is believed to act as a general repressor of transcription. To investigate in vivo the role of this protein in transcription regulation during development of a multicellular organism, we made transgenic tobacco plants that overexpress the gene for Arabidopsis histone H1. In all plants that overexpressed H1 the total H1-to-DNA ratio in chromatin increased 2.3-2.8 times compared with the physiological level. This was accompanied by 50-100% decrease of native tobacco H1. The phenotypic changes in H1-overexpressing plants ranged from mild to severe perturbations in morphological appearance and flowering. No correlation was observed between the extent of phenotypic change and the variation in the amount of overexpressed H1 or the presence or absence of the native tobacco H1. However, the severe phenotypic changes were correlated with early occurrence during plant growth of cells with abnormally heterochromatinized nuclei. Such cells occurred considerably later in plants with milder changes. Surprisingly, the ability of cells with highly heterochromatinized nuclei to fulfill basic physiological functions, including differentiation, was not markedly hampered. The results support the suggestion that chromatin structural changes dependent on H1 stoichiometry and on the profile of major H1 variants have limited regulatory effect on the activity of genes that control basal cellular functions. However, the H1-mediated chromatin changes can be of much greater importance for the regulation of genes involved in control of specific developmental programs.
Structural recovery in lesioned adult mammalian spinal cord by x-irradiation of the lesion site.
Authors: Kalderon N , Fuks Z
Published: July 2002

Mechanical injury to the adult mammalian spinal cord results in permanent morphological disintegration including severance/laceration of brain-cord axons at the lesion site. We report here that some of the structural consequences of injury can be averted by altering the cellular components of the lesion site with x-irradiation. We observed that localized irradiation of the unilaterally transected adult rat spinal cord when delivered during a defined time-window (third week) postinjury prevented cavitation, enabled establishment of structural integrity, and resulted in regrowth of severed corticospinal axons through the lesion site and into the distal stump. In addition, we examined the natural course of degeneration and cavitation at the site of lesion with time after injury, noting that through the third week postinjury recovery processes are in progress and only at the fourth week do the destructive processes take over. Our data suggest that the adult mammalian spinal cord has innate mechanisms required for recovery from injury and that timed intervention in certain cellular events by x-irradiation prevents the onset of degeneration and thus enables structural regenerative processes to proceed unhindered. We postulate that a radiation-sensitive subgroup of cells triggers the delayed degenerative processes. The identity of these intrusive cells and the mechanisms for triggering tissue degeneration are still unknown.
Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.
Authors: Wijmenga C , Gregory P E , Hajra A , Schröck E , Ried T , Eils R , Liu P P , Collins F S
Published: July 2002

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.
DUB-1, a deubiquitinating enzyme with growth-suppressing activity.
Authors: Zhu Y , Carroll M , Papa F R , Hochstrasser M , D'Andrea A D
Published: July 2002

Cytokines regulate cell growth by inducing the expression of specific target genes. Using the differential display method, we have cloned a cytokine-inducible immediate early gene, DUB-1 (for deubiquitinating enzyme). DUB-1 is related to members of the UBP superfamily of deubiquitinating enzymes, which includes the oncoprotein Tre-2. A glutathione S-transferase-DUB-1 fusion protein cleaved ubiquitin from a ubiquitin-beta-galactosidase protein. When a conserved cysteine residue of DUB-1, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. Continuous expression of DUB-1 from a steroid-inducible promoter induced growth arrest in the G1 phase of the cell cycle. Cells arrested by DUB-1 expression remained viable and resumed proliferation upon steroid withdrawal. Our results suggest that DUB-1 regulates cellular growth by modulating either the ubiquitin-dependent proteolysis or the ubiquitination state of an unknown growth regulatory factor(s).
A novel subviral agent associated with a geminivirus: The first report of a DNA satellite
Authors: Dry Ian B. , Krake Leslie R. , Rigden Justin E. , Rezaian M. Ali
Published: July 2002

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem–loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication.
Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids
Authors: Kenyon Lesley , Moraes Carlos T.
Published: July 2002

The nuclear and mitochondrial genomes coevolve to optimize approximately 100 different interactions necessary for an efficient ATP-generating system. This coevolution led to a species-specific compatibility between these genomes. We introduced mitochondrial DNA (mtDNA) from different primates into mtDNA-less human cells and selected for growth of cells with a functional oxidative phosphorylation system. mtDNA from common chimpanzee, pigmy chimpanzee, and gorilla were able to restore oxidative phosphorylation in the context of a human nuclear background, whereas mtDNA from orangutan, and species representative of Old-World monkeys, New-World monkeys, and lemurs were not. Oxygen consumption, a sensitive index of respiratory function, showed that mtDNA from chimpanzee, pigmy chimpanzee, and gorilla replaced the human mtDNA and restored respiration to essentially normal levels. Mitochondrial protein synthesis was also unaltered in successful “xenomitochondrial cybrids.” The abrupt failure of mtDNA from primate species that diverged from humans as recently as 8–18 million years ago to functionally replace human mtDNA suggests the presence of one or a few mutations affecting critical nuclear–mitochondrial genome interactions between these species. These cellular systems provide a demonstration of intergenus mtDNA transfer, expand more than 20-fold the number of mtDNA polymorphisms that can be analyzed in a human nuclear background, and provide a novel model for the study of nuclear–mitochondrial interactions.
Conformational fluctuations in single DNA molecules
Authors: Wennmalm Stefan , Edman Lars , Rigler Rudolf
Published: July 2002

Measurement of fluorescent lifetimes of dye-tagged DNA molecules reveal the existence of different conformations. Conformational fluctuations observed by fluorescence correlation spectroscopy give rise to a relaxation behavior that is described by “stretched” exponentials and indicates the presence of a distribution of transition rates between two conformations. Whether this is an inhomogeneous distribution, where each molecule contributes with its own reaction rate to the overall distribution, or a homogeneous distribution, where the reaction rate of each molecule is time-dependent, is not yet known. We used a tetramethylrhodamine-linked 217-bp DNA oligonucleotide as a probe for conformational fluctuations. Fluorescence fluctuations from single DNA molecules attached to a streptavidin-coated surface directly show the transitions between two conformational states. The conformational fluctuations typical for single molecules are similar to those seen in single ion channels in cell membranes.
The major chromatin protein histone H1 binds preferentially to
cis
-platinum-damaged DNA
Authors: Yaneva Julia , Leuba Sanford H. , van Holde Kensal , Zlatanova Jordanka
Published: July 2002

Both
cis-
diamminedichloroplatinum(II) (cisplatin or
cis
-DDP) and
trans
-diamminedichloroplatinum(II) form covalent adducts with DNA. However, only the
cis
isomer is a potent anticancer agent. It has been postulated that the selective action of
cis
-DDP occurs through specific binding of nuclear proteins to
cis
-DDP-damaged DNA sites and that binding blocks DNA repair. We find that a very abundant nuclear protein, the linker histone H1, binds much more strongly to cis-platinated DNA than to trans-platinated or unmodified DNA. In competition experiments, H1 is shown to bind much more strongly than HMG1, which had been previously considered a major candidate for such binding
in vivo
.
Functional differentiation of T cells in the intestine of T cell receptor transgenic mice
Authors: Hurst Stephen D. , Sitterding Stephanie M. , Ji Sandy , Barrett Terrence A.
Published: July 2002

The intestinal lamina propria (LP) is a major effector site of the mucosal immune system where antigen-specific and antigen-nonspecific factors shape the functional responses of CD4+T helper cells. To study the functional differentiation of LP T helper cells we utilized DO11.10 T cell receptor (TCR) transgenic (Tg) mice that expressed a clonotypic TCR specific for a class II major histocompatibility complex-restricted peptide of chicken ovalbumin. The majority of cells expressing Tg TCR (Tg+) in peripheral lymphoid tissue expressed naive surface phenotypes whereas nearly all Tg+T cells in the intestinal LP expressed an activated/memory-like phenotype. Flow cytometric analysis of Tg+T cell populations revealed that a small proportion of cells in peripheral lymphoid tissue but nearly all cells in the LP expressed dual (Tg plus non-Tg) TCRs. In Tg × recombinase-activating-gene-1-deficient (Tg ×RAG-1−/−) mice, splenic and LP T cells expressed naive surface phenotypes and produced cytokines equivalent to naive splenic cells from Tg ×RAG-1+/+mice. In contrast, Tg LP cells from Tg ×RAG-1+/+mice produced 35-fold greater levels of interferon-γ and 5-fold greater levels of interleukin 4 compared with naive splenic cells. These findings suggested that activation of Tg+T cells through endogenous non-Tg TCR had promoted the localization and differentiation of memory-like effector T helper cells in the intestine.
The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase
Authors: de Nadal Eulàlia , Clotet Josep , Posas Francesc , Serrano Ramón , Gomez Néstor , Ariño Joaquín
Published: July 2002

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of
ENA1
, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of
HAL3
(also known as
SIS2
) has no effect on salt tolerance in the absence of
PPZ1
, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione
S
-transferase, and it also copurifies with affinity-purified glutathione
S
-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed.
In vitro
experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a
slt2/mpk1
mitogen-activated protein kinase mutant (thus mimicking the deletion of
PPZ1
). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.
Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway
Authors: Scott Pamela H. , Brunn Gregory J. , Kohn Aimee D. , Roth Richard A. , Lawrence John C.
Published: July 2002

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the
32
P content of mTOR that was determined after purifying the protein from
32
P-labeled adipocytes with rapamycin⋅FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.
A nod factor binding lectin with apyrase activity from legume roots
Authors: Etzler Marilynn E. , Kalsi Gurpreet , Ewing Nicholas N. , Roberts Nicholas J. , Day R. Bradley , Murphy Judith B.
Published: July 2002

A lectin isolated from the roots of the legume,Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date. This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands. Thislectin–nucleotidephosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes. LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia. These properties suggest that this protein may play a role in the rhizobium–legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant.
Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogenPseudomonas syringaepathovar phaseolicola
Authors: Jackson Robert W. , Athanassopoulos Evangelos , Tsiamis George , Mansfield John W. , Sesma Ane , Arnold Dawn L. , Gibbon Marjorie J. , Murillo Jesus , Taylor John D. , Vivian Alan
Published: July 2002

The 154-kb plasmid was cured from race 7 strain 1449B of the phytopathogenPseudomonas syringaepv. phaseolicola (Pph). Cured strains lost virulence toward bean, causing the hypersensitive reaction in previously susceptible cultivars. Restoration of virulence was achieved by complementation with cosmid clones spanning a 30-kb region of the plasmid that contained previously identified avirulence (avr) genesavrD,avrPphC, andavrPphF. Single transposon insertions at multiple sites (including one located inavrPphF) abolished restoration of virulence by genomic clones. Sequencing 11 kb of the complementing region identified three potential virulence (vir) genes that were predicted to encode hydrophilic proteins and shared thehrp-box promoter motif indicating regulation by HrpL. One gene achieved partial restoration of virulence when cloned on its own and therefore was designatedvirPphAas the first (A) gene fromPphto be identified for virulence function. In soybean,virPphAacted as anavrgene controlling expression of a rapid cultivar-specific hypersensitive reaction. Sequencing also revealed the presence of homologs of the insertion sequence IS100fromYersiniaand transposase Tn501fromP. aeruginosa. The proximity of severalavrandvirgenes together with mobile elements, as well as G+C content significantly lower than that expected forP. syringae, indicates that we have located a plasmid-borne pathogenicity island equivalent to those found in mammalian pathogens.
Verbal and Spatial Short‐term Memory: Two Sources of Developmental Evidence Consistent with Common Underlying Processes
Authors: Chuah Y. M. Lisa , Maybery Murray T.
Published: July 2002

Developments in serial short‐term memory (STM) typically have been assessed using verbal items, and mechanisms of development unique to the verbal system have been postulated. Here we review two studies of the development of spatial STM. The first (Chuah & Maybery, 1999) showed that variance in spatial span was partitioned among a set of predictor variables in a configuration very similar to that found for verbal span. The second (Maybery & Chuah, 1999) examined the timing of recall for spatial sequences. Intervals between touches as children recalled spatial sequences showed comparable effects to those reported for the oral recall of verbal sequences. Discussion focuses on processes that could serve both verbal and spatial memory and mediate span development.
Gender, Memory, and National Myths: Ol'ga Berggol'ts and the Siege of Leningrad
Authors: Kirschenbaum Lisa
Published: July 2002

In Russia, the memory of the Second World War has been at once deeply personal and profoundly political. Largely erased from official memory until Stalin's death, the story of the war became, in the 1960s, a key means of legitimizing the Soviet state. The mythic “20 million”—more recent estimates are closer to 30 million war dead—became the heart of a lasting and state-sanctioned collective memory of shared suffering, patriotism, and redemption. As historian Nina Tumarkin has argued, the official “cult” of the war began to crumble in the mid-1980s, and what she calls “raw human memory,” personal stories untainted by the myth created from above, began to emerge. Tumarkin contends that the “winds of glasnost' and perestroika” effectively “ravaged” both the state-sanctioned “myth” and the “shared memory” of the Great Patriotic War. Personal tragedies began to replace the official tale of national triumph.
The Influence of only on Syntactic processing of “Long” Relative Clause Sentences
Authors: Liversedge Simon P. , Paterson Kevin B. , Clayes Emma L.
Published: July 2002

We report an eye movement experiment investigating the influence of the focus operator only on syntactic processing of “long” relative clause sentences. Paterson, Liversedge, and Underwood (1999) found that readers were garden pathed by “short” reduced relative clause sentences containing the focus operator only. They argued that due to thematic differences between “short” and “long” relative clause sentences, garden path effect might not occur when “long” reduced relative clause sentences are read. Eye-tracking data show that garden path effects found during initial processing of the disambiguating verb of “long” reduced sentences without only were absent or delayed in the case of counterparts with only. We discuss our results in terms of current theories of sentence processing.
Attention‐deficit/hyperactivity disorder in the drug and alcohol clinic
Authors: Tucker Peter
Published: July 2002

Attention‐deficit/hyperactivity disorder (ADHD), a disorder generally seen in children but more recently found in many cases to persist to adulthood, has been found to be associated with later development of substance abuse. The author reviews evidence for this, and for the possible mediating role of antisocial behaviour in this association. It is concluded that the latter may have a predominating role, but that in any case there is likely to be a significant number of adults with ADHD in substance‐abusing populations. It is doubtful that such individuals are truly self‐medicating their psychiatric disorder. Suggestions are made regarding an approach to this problem of dual diagnosis.
Regional culture in post-war Friuli: Literature in dialect, nationalism and friulanità
Authors: Johnson John R. L.
Published: July 2002

The unprecedented increase in literary production in Friuli in the post-war period has coincided with the rise of popular ethno-nationalism in the region. Although there is an evident connection between the political, social and cultural fields in Friuli, this relationship is both complex and full of potential conflicts. This paper provides a brief overview of Friulian regionalism, before considering the specific role assigned to literature in Friulian by proponents of regional autonomy. It examines the problematic nature of the dominant ideology of friulanità and discusses the responses of a number of authors to the prevailing themes of cultural discourse in the region. In conclusion, it examines the ideological conflicts caused by modernisation in the region, and considers the impact that the transformation of the region has had on the literary debate, concentrating on the difficulties caused by Friulian linguistic purism.
Nicotine dependency of a girl at the age of 18 months
Authors: Sir Aytekin , ÖZkan Mustafa
Published: July 2002

This case report describes severe nicotine dependency developing at the age of 18 months. Familial, genetic, biological aspects of the case and the therapeutic strategy are discussed.
The effect of ethanol and acetaldehyde on microsomal and mitochondrial membrane fatty acid profiles in cultured rat astroglia
Authors: HOLOWNIA A. , MESKAR A. , MENEZ J. F. , LEDIG M. , BRASZKO J. J.
Published: July 2002

It has been shown that free radical damage may be involved in ethanol‐induced cytotoxicity in cultured neural cells. Since changes in oxidative metabolism and the resulting lipid peroxidation readily modify biological membranes and alter cell functions we studied the effect of ethanol and its metabolite acetaldehyde on rat astroglial fatty acids profiles in the most common lipid classes of mitochondrial and microsomal membranes, i.e. phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Rat astroglial cells were grown for 1 week in the presence of 50 m M or 100 m M ethanol. To examine acetaldehyde effects we used a 4‐day co‐culture model consisting of astroglial cells and alcohol dehydrogenase‐transfected Chinese hamster ovary (CHO) cells. Acetaldehyde produced by these cells reached 172 mu M and 265 mu M, respectively, for ethanol concentrations of 10 and 20 m M. Mitochondrial and microsomal membranes were prepared by differential centrifugation, phosphatidylcholine and phosphatidylethanolamine were separated using thin layer chromatography and fatty acid quantitation was performed by GLC. Neither ethanol nor acetaldehyde changed the mitochondrial phosphatidylcholine or phosphatidylethanolamine profiles of total saturated, mono‐unsaturated or polyunsaturated fatty acids. However, some significant alterations in particular fatty acids appeared especially after acetaldehyde but also after the highest ethanol dose. In microsomal phosphatidylcholine monounsaturated fatty acids were significantly increased after both, ethanol and acetaldehyde exposure. Among polyunsaturated fatty acids, arachidonic acid was found to be especially affected by both ethanol and acetaldehyde. Similar decreases were observed in adrenic, docosapentaenoic and docosahexaenoic acids in the groups treated with ethanol. In microsomal phosphatidylethanolamine, ethanol and acetaldehyde decreased monounsaturated and some polyunsaturated fatty acids. These data support the role of peroxidative processes in cultured rat astroglia exposed to ethanol and point to the role of acetaldehyde in this mechanism.
The prevalence of alcohol misuse in patients undergoing cataract surgery
Authors: DAVIS A. R. , WILKINS M. , DEW T. , SHERWOOD R. , COAKES R. , PETERS T. J.
Published: July 2002

If a factor could be identified which delayed the onset of cataract by 10 years, the number of annual cataract operations worldwide has been estimated to decrease by 45%. A case‐control study compared alcohol consumption in 78 patients attending for routine cataract surgery in South East London with data from a large population‐based survey. Male cataract patients had a significantly greater risk of being harmful drinkers (odds ratio = 8, p = 0.007) than the controls. The harmful male drinkers were significantly younger than the non‐drinkers with cataract (mean difference 15 years, p < 0.007). Female cataract patients were not more likely to be excessive drinkers than controls. The female drinkers with cataract were of a similar age to the non‐drinking female patients with cataract. Haematological and biochemical indices of alcohol toxicity indicated five patients who were likely to be harmful drinkers, but who had denied this on direct questioning. Seven (26%) of the male patients had a low serum 25 hydroxycholecalciferol although the levels were normal in the female patients. These results support the view that excess alcohol consumption is related to cataract formation and suggest that alcohol causes premature cataract formation in male, but not female patients. Alcohol consumption is amenable to intervention and suggests that such intervention could have a significant impact on the need for cataract surgery.
Tryptophan pyrrolase gene expression in an alcohol preferring and non‐preferring mouse strain
Authors: ORETTI R. G. , O'DONOVAN M. C. , McGUFFIN P. , BUCKLAND P.
Published: July 2002

There is considerable evidence that the level of cerebral serotonin plays a key role in the volitional consumption of ethanol in both man and animals. Naive alcohol‐preferring C57BL/6J mice have been shown to have a lower cerebral serotonin content compared to the non‐preferring CBA/Ca mouse strain. This has been attributed to the enhancement of hepatic tryptophan pyrrolase activity in C57 mice. Activity and/or expression of tryptophan pyrrolase may be an important biological determinant of alcohol preference. We have investigated the possible mechanism/s underlying this strain difference in tryptophan pyrrolase activity by assaying both mRNA levels encoding for the tryptophan pyrrolase gene and by mutational analysis of tryptophan pyrrolase cDNA. We were unable to demonstrate any difference in tryptophan pyrrolase mRNA levels between naive C57 and CBA mice. Tryptophan pyrrolase mRNA levels were increased following starvation in C57 mice and following glucocorticoid administration in both C57 and CBA mice. Heteroduplex mutational analysis failed to detect any tryptophan pyrrolase cDNA sequence heterogeneity between these mice strains.
Biological markers as indicators for relapse in alcohol‐dependent patients
Authors: MUNDLE GÖTZ , ACKERMANN KLAUS , MANN KARL
Published: July 2002

Although biological markers such as carbohydrate‐deficient transferrin (CDT), γ‐glutamyl transferase (GGT) and mean corpuscular volume (MCV) have been used as indicators for heavy alcohol consumption and alcoholism little information is available on the utitlity of these markers in detecting relapses. In this study the value of the biological markers CDT, GGT and MCV was examined in monitoring an outpatient treatment programme for alcohol‐dependent patients. In 163 male alcoholic patients CDT, GGT and mean corpuscular volume (MCV) were assayed at the beginning and after 6 months during the outpatient programme. All markers distingushed between relapsers and abstainers (p<0.01). The sensitivity for relapses was 55% for CDT, 50% for GGT and 20% for MCV. Combining all markers the sensitivity could be enhanced to 85%, with only a little loss of specificity (85%). The highest positive predictive value was 73% for CDT used as a single marker. The negative predictive value (CDT 93%, GGT 92%, MCV 88%) and the diagnostic efficiency (CDT 91%, GGT 87%, MCV 85%) of all markers were very high. These results indicate that CDT is the most efficient marker for alcohol relapses, followed by GGT. MCV seems to be a marker of second choice.
Oxidation of acetaldehyde by isolated aortic rings of UChA and UChB rats
Authors: QUINTANILLA MARÍA ELENA , TAMPIER LUTSKE
Published: July 2002

The rate of acetaldehyde metabolism was measured in aortic rings from rat strains genetically bred for high (UChB) and low (UChA) voluntary ethanol consumption. The results show that in aortic rings from naive UChB rats, acetaldehyde oxidation rates were significantly greater than the rates observed in aortic rings from naive UChA rats. These strain differences are explained by different activity of vascular low‐Km aldehyde dehydrogenase (ALDH). Chronic feeding of ethanol to UChA rats did not alter their aortic ALDH activity. The results of the present study provides additional evidence that the activity variation for the low‐Km ALDH between both rat strains exists in various organs and tissues.
Oligomerization-Dependent Regulation of Motility and Morphogenesis by the Collagen Xviii Nc1/Endostatin Domain
Authors: Kuo Calvin J. , LaMontagne Kenneth R. , Garcia-Cardeña Guillermo , Ackley Brian D. , Kalman Daniel , Park Susan , Christofferson Rolf , Kamihara Junne , Ding Yuan-Hua , Lo Kin-Ming , Gillies Stephen , Folkman Judah , Mulligan Richard C. , Javaherian Kashi
Published: July 2002

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase–stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.
Differentiation- and stress-dependent nuclear cytoplasmic redistribution of myopodin, a novel actin-bundling protein
Authors: Weins Astrid , Schwarz Karin , Faul Christian , Barisoni Laura , Linke Wolfgang A. , Mundel Peter
Published: July 2002

We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with α-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A–sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein–myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.
T Cell Receptor Signals Enhance Susceptibility to Fas-mediated Apoptosis
Authors: Wong Brian , Arron Joseph , Choi Yongwon
Published: July 2002

Fas(CD95) and its ligand (FasL) interaction plays a pivotal role in T cell receptor (TCR)-mediated apoptosis. However, the susceptibility of T cells to Fas-mediated apoptosis is tightly regulated during immune responses, a regulation which is thought to maintain the antigen-specificity of T cell apoptosis. Here we show that TCR stimulation enhances the induction of Fas-mediated apoptosis. In addition, using a mutant T cell hybridoma with impaired FasL expression, we show that the synergy provided by TCR stimulation can be mimicked by activators of PKC but not calcium influx. This effect cannot be inhibited by actinomycin D, suggesting that TCR stimulation leads to the alteration in preexisting signaling molecules to enhance Fas-mediated apoptosis. Our results therefore provide a mechanism of how Fas-FasL interactions lead to T cell death in an antigen-specific manner via repetitive antigen stimulation.
Novel Mutants Define Genes Required for the Expression of Human Histocompatibility Leukocyte Antigen DM: Evidence for Loci on Human Chromosome 6p
Authors: Fling Steven P. , Rak Jennifer , Muczynski Kimberly A. , Arp Benjamin , Pious Donald
Published: July 2002

We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II–associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II–peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM null mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II–restricted antigen processing.
Functional Dichotomy in Natural Killer Cell Signaling
Authors: Colucci Francesco , Rosmaraki Eleftheria , Bregenholt Søren , Samson Sandrine I. , Di Bartolo Vincenzo , Turner Martin , Vanes Lesley , Tybulewicz Victor , Di Santo James P.
Published: July 2002

The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen–receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1−/− mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1−/− mice produced normal amounts of interferon (IFN)-γ in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1−/− NK cells resulted in normal IFN-γ production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1−/− NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-γ production.
Bcl-2 Cooperates with Promyelocytic Leukemia Retinoic Acid Receptor α Chimeric Protein (Pmlrarα) to Block Neutrophil Differentiation and Initiate Acute Leukemia
Authors: Kogan Scott C. , Brown Diane E. , Shultz David B. , Truong Bao-Tran H. , Lallemand-Breitenbach Valerie , Guillemin Marie-Claude , Lagasse Eric , Weissman Irving L. , Bishop J. Michael
Published: July 2002

The promyelocytic leukemia retinoic acid receptor α (PMLRARα) chimeric protein is associated with acute promyelocytic leukemia (APL). PMLRARα transgenic mice develop leukemia only after several months, suggesting that PMLRARα does not by itself confer a fully malignant phenotype. Suppression of apoptosis can have a central role in tumorigenesis; therefore, we assessed whether BCL-2 influenced the ability of PMLRARα to initiate leukemia. Evaluation of preleukemic animals showed that whereas PMLRARα alone modestly altered neutrophil maturation, the combination of PMLRARα and BCL-2 caused a marked accumulation of immature myeloid cells in bone marrow. Leukemias developed more rapidly in mice coexpressing PMLRARα and BCL-2 than in mice expressing PMLRARα alone, and all mice expressing both transgenes succumbed to leukemia by 7 mo. Although both preleukemic, doubly transgenic mice and leukemic animals had abundant promyelocytes in the bone marrow, only leukemic mice exhibited thrombocytopenia and dissemination of immature cells. Recurrent gain of chromosomes 7, 8, 10, and 15 and recurrent loss of chromosome 2 were identified in the leukemias. These chromosomal changes may be responsible for the suppression of normal hematopoiesis and dissemination characteristic of the acute leukemias. Our results indicate that genetic changes that inhibit apoptosis can cooperate with PMLRARα to initiate APL.
Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus
Authors: Bevensee Mark O. , Apkon Michael , Boron Walter F.
Published: July 2002

In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na+-driven influx of HCO3− causes the increase in intracellular pH (pHi) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO2/17 mM HCO3−. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na+-driven Cl-HCO3 exchanger, an electrogenic Na/HCO3 cotransporter, or an electroneutral Na/HCO3 cotransporter. To determine if the transporter is a Na+-driven Cl-HCO3 exchanger, we depleted the cells of intracellular Cl− by incubating them in a Cl−-free solution for an average of ∼11 min. We verified the depletion with the Cl−-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl−-depleted cells, the pHi still increases after one or more exposures to CO2/HCO3−. Furthermore, the pHi decrease elicited by external Na+ removal does not require external Cl−. Therefore, the transporter cannot be a Na+-driven Cl-HCO3 exchanger. To determine if the transporter is an electrogenic Na/ HCO3 cotransporter, we measured pHi and plasma membrane voltage (Vm) while removing external Na+, in the presence/absence of CO2/HCO3− and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO2/HCO3− solutions contained 20% CO2 and 68 mM HCO3−, pH 7.3, to maximize the HCO3− flux. In pHi experiments, removing external Na+ in the presence of CO2/HCO3− elicited an equivalent HCO3− efflux of 281 μM s−1. The HCO3− influx elicited by returning external Na+ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive Vm change was 3.3 mV. Indeed, we found that removing external Na+ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO2/ HCO3−. Thus, the Na/HCO3 cotransporter is electrogenic. Because a cotransporter with a Na+:HCO3− stoichiometry of 1:3 or higher would predict a net HCO3− efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO3 cotransporter with a stoichiometry of 1:2.
Mating behaviours of Daphnia pulicaria, a cyclic parthenogen: comparisons with copepods
Authors: Brewer Matthew C.
Published: July 2002

The pre–and post–contact mating behaviours ofDaphnia pulicariaare investigated by direct observations of vertical distributions, swimming behaviours and male–female interactions. Analysis of vertical distributions in a 1 m deep, thermally stratified migration chamber reveals that females were always located in the upper layer of the water column but males exhibited a bimodal depth distribution, in which an individual's depth was a function of body length and water temperature. The observed distributions of males may be the result of several interacting pressures; predation avoidance, life–history optimization, and avoidance of assortative mating. Male swimming behaviour was faster and orthogonal to that of females, which is in agreement with the predictions of encounter–rate maximization models. Video recordings of males and females interacting in a 1–litre vessel showed that males both pursued and contacted other males more often than females. Thus, there was no evidence thatDaphniaare able to use water–borne chemical signals to locate and identify potential mates. However, the average duration of male–female contacts (13.8 s) was much longer than those between males (1.6 s), suggesting that males can determine the sex of contacted individuals.Daphniamating behaviour is significantly more complex than previously acknowledged. In contrast to the conventional view ofDaphniamales swimming more–or–less randomly and mating with any individual encountered, they exhibit behaviours which increase the potential of mating with females while reducing the risk of predation. Several male behaviours, such as ‘scanning’ and the performance of area–restricted spirals upon encounter, are similar to those reported for some copepods and may be common to zooplankton that lack sophisticated chemosensory abilities. The possibility thatDaphniamay also be able to assess such important female attributes as species and reproductive status is discussed.