bluuebunny/crossref_metadata_2025 huggingface dataset:
A cloning method to identify caspases and their regulators in yeast: Identification of
Drosophila
IAP1 as an inhibitor of the
Drosophila
caspase DCP-1
Authors: Hawkins Christine J. , Wang Susan L. , Hay Bruce A.
Published: July 2002

Site-specific proteases play critical roles in regulating many cellular processes. To identify novel site-specific proteases, their regulators, and substrates, we have designed a general reporter system in
Saccharomyces cerevisiae
in which a transcription factor is linked to the intracellular domain of a transmembrane protein by protease cleavage sites. Here, we explore the efficacy of this approach by using caspases, a family of aspartate-specific cysteine proteases, as a model. Introduction of an active caspase into cells that express a caspase-cleavable reporter results in the release of the transcription factor from the membrane and subsequent activation of a nuclear reporter. We show that known caspases activate the reporter, that an activator of caspase activity stimulates reporter activation in the presence of an otherwise inactive caspase, and that caspase inhibitors suppress caspase-dependent reporter activity. We also find that, although low or moderate levels of active caspase expression do not compromise yeast cell growth, higher level expression leads to lethality. We have exploited this observation to isolate clones from a
Drosophila
embryo cDNA library that block DCP-1 caspase-dependent yeast cell death. Among these clones, we identified the known cell death inhibitor DIAP1. We showed, by using bacterially synthesized proteins, that glutathione
S
-transferase–DIAP1 directly inhibits DCP-1 caspase activity but that it had minimal effect on the activity of a predomainless version of a second
Drosophila
caspase, drICE.
Microsatellite instability in
Drosophila spellchecker1
(MutS homolog) mutants
Authors: Flores Carlos , Engels William
Published: July 2002

We have cloned a
mutS
homolog from
Drosophila melanogaster
called
spellchecker1 (spel1
) and have constructed
spel1
mutant flies. MutS proteins promote the correction of DNA mismatches and serve important roles in DNA replication, recombination, and repair. The
spel1
gene belongs to a subfamily of
mutS
first characterized by the MSH2 gene of yeast and which also includes
hMSH2,
one of the two major hereditary nonpolyposis colon cancer loci of humans. Like
msh2
mutants in other species, we find that flies lacking the
spel1
gene suffer a highly increased rate of instability in long runs of dinucleotide repeats when analyzed after 10–12 fly generations. Using a new assay, we have also discovered that mutations in
spel1
decrease the stability of a dinucleotide repeat when it is copied into the site of a double-strand break during gene conversion. Contrary to the case in mammalian cells,
spel1
deficiency does not affect tolerance of flies to a methylating agent nor does it affect resistance to γ-irradiation.
The p42 variant of ETS1 protein rescues defective Fas-induced apoptosis in colon carcinoma cells
Authors: Li Runzhao , Pei Huiping , Papas Takis
Published: July 2002

ETS1 is a cellular homologue of the product of the viral
ets
oncogene of the E26 virus, and it functions as a tissue-specific transcription factor. It plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. ETS1 controls the expression of critical genes involved in these processes by binding to
ets
binding sites present in the transcriptional regulatory regions. The
ETS1
gene generates two proteins, p51 and a spliced variant, p42, lacking exon VII. In this paper we show that p42-ETS1 expression bypasses the damaged Fas-induced apoptotic pathway in DLD1 colon carcinoma cells by up-regulating interleukin 1β-converting enzyme (ICE)/caspase-1 and causes these cancer cells to become susceptible to the effects of the normal apoptosis activation system. ICE/caspase-1 is a redundant system in many cells and tissues, and here we demonstrate that it is important in activating apoptosis in cells where the normal apoptosis pathway is blocked. Blocking ICE/caspase-1 activity by using specific inhibitors of this protease prevents the p42-ETS1-induced apoptosis from occurring, indicating that the induced ICE/caspase-1 enzyme is responsible for killing the cancer cells. p42-ETS1 activates a critical alternative apoptosis pathway in cancer cells that are resistant to normal immune attack, and thus it may be useful as an anticancer therapeutic.
A logical analysis of T cell activation and anergy
Authors: Kaufman Marcelle , Andris Fabienne , Leo Oberdan
Published: July 2002

Interaction of the antigen-specific receptor of T lymphocytes with its antigenic ligand can lead either to cell activation or to a state of profound unresponsiveness (anergy). Although subtle changes in the nature of the ligand or of the antigen-presenting cell have been shown to affect the outcome of T cell receptor ligation, the mechanism by which the same receptor can induce alternative cellular responses is not completely understood. A model for explaining both positive (cell proliferation and cytokine production) and negative (anergy induction) signaling of T lymphocytes is described herein. This model relies on the autophosphorylative properties of the tyrosine kinases associated with the T cell receptor. One of its basic assumptions is that the kinase activity of these receptor-associated enzymes remains above background level after ligand removal and is responsible for cellular unresponsiveness. Using a simple Boolean formalism, we show how the timing of the binding and intracellular signal-transduction events can affect the properties of receptor signaling and determine the type of cellular response. The present approach integrates into a common framework a large body of experimental observations and allows specification of conditions leading to cellular activation or to anergy.
Pathways of As(III) detoxification in
Saccharomyces cerevisiae
Authors: Ghosh Mallika , Shen Jian , Rosen Barry P.
Published: July 2002

Saccharomyces cerevisiae
has two independent transport systems for the removal of arsenite from the cytosol. Acr3p is a plasma membrane transporter that confers resistance to arsenite, presumably by arsenite extrusion from the cells. Ycf1p, a member of the ABC transporter superfamily, catalyzes the ATP-driven uptake of As(III) into the vacuole, also producing resistance to arsenite. Vacuolar accumulation requires a reductant such as glutathione, suggesting that the substrate is the glutathione conjugate, As(GS)
3
. Disruption of either the
ACR3
or
YCF1
gene results in sensitivity to arsenite and disruption of both genes produces additive hypersensitivity. Thus, Acr3p and Ycf1p represent separate pathways for the detoxification of arsenite in yeast.
Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy
Authors: Kunisada Keita , Negoro Shinji , Tone Eiroh , Funamoto Masanobu , Osugi Tomoaki , Yamada Shuichi , Okabe Masaru , Kishimoto Tadamitsu , Yamauchi-Takihara Keiko
Published: July 2002

The signal transducer and activator of transcription (STAT) 3, a transcriptional factor downstream of several cytokines, is activated by Janus kinase families and plays a pivotal role in cardiac hypertrophy through gp130. To determine the physiological significance of STAT3in vivo, transgenic mice with cardiac-specific overexpression of theStat3gene (STAT3-TG) were generated. STAT3-TG manifested myocardial hypertrophy at 12 wk of age with increased expression of theatrial natriuretic factor(ANF),β-myosin heavy chain(MHC), andcardiotrophin(CT)-1genes. The animals were injected i.p. with 15 mg/kg doxorubicin (Dox), an antineoplastic drug with restricted use because of its cardiotoxicity. The survival rates after 10 days were 25% (5/20) for control littermates (WT), but 80% (16/20) for STAT3-TG (P< 0.01). WT showed increased expression of β-MHC and ANF mRNAs in the hearts 1 day after Dox treatment; this expression peaked at 3 days, suggesting that the WT suffered from congestive heart failure. Although the expression of these mRNAs was elevated in STAT3-TG hearts before Dox treatment, no additional increase was observed after the treatment. Dox administration significantly reduced the expression of thecardiacα-actinandStat3genes in WT hearts but not in STAT3-TG. These results provide direct evidence that STAT3 transduces not only a hypertrophic signal but also a protective signal against Dox-induced cardiomyopathy by inhibiting reduction of cardiac contractile genes and inducing cardiac protective factors.
Asparagine-proline sequence within membrane-spanning segment of SREBP triggers intramembrane cleavage by Site-2 protease
Authors: Ye Jin , Davé Utpal P. , Grishin Nick V. , Goldstein Joseph L. , Brown Michael S.
Published: July 2002

The NH
2
-terminal domains of membrane-bound sterol regulatory element-binding proteins (SREBPs) are released into the cytosol by regulated intramembrane proteolysis, after which they enter the nucleus to activate genes encoding lipid biosynthetic enzymes. Intramembrane proteolysis is catalyzed by Site-2 protease (S2P), a hydrophobic zinc metalloprotease that cleaves SREBPs at a membrane-embedded leucine-cysteine bond. In the current study, we use domain-swapping methods to localize the residues within the SREBP-2 membrane-spanning segment that are required for cleavage by S2P. The studies reveal a requirement for an asparagine-proline sequence in the middle third of the transmembrane segment. We propose a model in which the asparagine-proline sequence serves as an NH
2
-terminal cap for a portion of the transmembrane α-helix of SREBP, allowing the remainder of the α-helix to unwind partially to expose the peptide bond for cleavage by S2P.
GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst
Authors: Carayannopoulos Mary O. , Chi Maggie M.-Y. , Cui Ying , Pingsterhaus Joyce M. , McKnight Robert A. , Mueckler Mike , Devaskar Sherin U. , Moley Kelle H.
Published: July 2002

Mammalian preimplantation blastocysts exhibit insulin-stimulated
glucose uptake despite the absence of the only known insulin-regulated
transporter, GLUT4. We describe a previously unidentified member of the
mammalian facilitative GLUT superfamily that exhibits ≈20–25%
identity with other murine facilitative GLUTs. Insulin induces a change
in the intracellular localization of this protein, which translates
into increased glucose uptake into the blastocyst, a process that is
inhibited by antisense oligoprobes. Presence of this transporter may be
necessary for successful blastocyst development, fuel metabolism, and
subsequent implantation. Moreover, the existence of an alternative
transporter may explain examples in other tissues of insulin-regulated
glucose transport in the absence of GLUT4.
Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2
Authors: Schubert Ulrich , Ott David E. , Chertova Elena N. , Welker Reinhold , Tessmer Uwe , Princiotta Michael F. , Bennink Jack R. , Kräusslich Hans-Georg , Yewdell Jonathan W.
Published: July 2002

Retrovirus assembly and maturation involve folding and transport of
viral proteins to the virus assembly site followed by subsequent
proteolytic cleavage of the Gag polyprotein within the nascent virion.
We report that inhibiting proteasomes severely decreases the budding,
maturation, and infectivity of HIV. Although processing of the Env
glycoproteins is not changed, proteasome inhibitors inhibit processing
of Gag polyprotein by the viral protease without affecting the activity
of the HIV-1 viral protease itself, as demonstrated by
in
vitro
processing of HIV-1 Gag polyprotein Pr55. Furthermore,
this effect occurs independently of the virus release function of the
HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome
inhibitors also reduce virus release and Gag processing of HIV-2.
Electron microscopy analysis revealed ultrastructural changes in
budding virions similar to mutants in the late assembly domain of
p6
gag
, a C-terminal domain of Pr55 required for efficient
virus maturation and release. Proteasome inhibition reduced the level
of free ubiquitin in HIV-1-infected cells and prevented
monoubiquitination of p6
gag
. Consistent with this, viruses
with mutations in PR or p6
gag
were resistant to detrimental
effects mediated by proteasome inhibitors. These results indicate the
requirement for an active proteasome/ubiquitin system in release and
maturation of infectious HIV particles and provide a potential
pharmaceutical strategy for interfering with retrovirus replication.
Mechanism for a transcriptional activator that works at the isomerization step
Authors: Dove Simon L. , Huang Franklin W. , Hochschild Ann
Published: July 2002

Transcriptional activators in prokaryotes have been shown to
stimulate different steps in the initiation process including the
initial binding of RNA polymerase (RNAP) to the promoter and a
postbinding step known as the isomerization step. Evidence suggests
that activators that affect initial binding can work by a cooperative
binding mechanism by making energetically favorable contacts with RNAP,
but the mechanism by which activators affect the isomerization step is
unclear. A well-studied example of an activator that normally exerts
its effect exclusively on the isomerization step is the bacteriophage
λ cI protein (λcI), which has been shown genetically to interact
with the C-terminal region of the σ
70
subunit of RNAP. We
show here that the interaction between λcI and σ can stimulate
transcription even when the relevant portion of σ is transplanted to
another subunit of RNAP. This activation depends on the ability of
λcI to stabilize the binding of the transplanted σ moiety to an
ectopic −35 element. Based on these and previous findings, we discuss
a simple model that explains how an activator's ability to stabilize
the binding of an RNAP subdomain to the DNA can account for its effect
on either the initial binding of RNAP to a promoter or the
isomerization step.
Peptide-in-groove interactions link target proteins to the β-propeller of clathrin
Authors: ter Haar Ernst , Harrison Stephen C. , Kirchhausen Tomas
Published: July 2002

The “WD40” domain is a widespread recognition module for linking partner proteins in intracellular networks of signaling and sorting. The clathrin amino-terminal domain, which directs incorporation of cargo into coated pits, is a β-propeller closely related in structure to WD40 modules. The crystallographically determined structures of complexes of the clathrin-terminal domain with peptides derived from two different cargo adaptors, β-arrestin 2 and the β-subunit of the AP-3 complex, reveal strikingly similar peptide-in-groove interactions. The two peptides in our structures contain related, five-residue motifs, which form the core of their contact with clathrin. A number of other proteins involved in endocytosis have similar “clathrin-box” motifs, and it therefore is likely that they all bind the terminal domain in the same way. We propose that a peptide-in-groove interaction is an important general mode by which β-propellers recognize specific target proteins.
Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster
Authors: Bai Chang , Connolly Brett , Metzker Michael L. , Hilliard Catherine A. , Liu Xiaomei , Sandig Volker , Soderman Avery , Galloway Sheila M. , Liu Qingyun , Austin Christopher P. , Caskey C. Thomas
Published: July 2002

Fas-mediated apoptosis is an important regulator of cell survival, and abnormalities in this system have been shown to result in a number of human pathological conditions. A secreted member of the tumor necrosis factor receptor superfamily, DcR3, was recently reported to be amplified in human lung and colon cancers as a negative regulator of Fas-mediated apoptosis. We identified this gene, which we call M68. M68 genomic DNA, mRNA, and protein levels were examined in a series of human gastrointestinal tract tumors. Using M68 immunohistochemistry and a scoring system similar to that used for HER-2/neu, we found that M68 protein was overexpressed in 30 of 68 (44%) human adenocarcinomas of the esophagus, stomach, colon, and rectum. Tumors examined by Northern blot revealed M68 mRNA highly elevated in a similar fraction of primary tumors from the same gastrointestinal tract regions, as well as in the colon adenocarcinoma cell lines SW480 and SW1116. Further, we found M68 protein to be overexpressed in a substantial number of tumors in which gene amplification could not be detected by fluorescence
in situ
hybridization or quantitative genomic PCR, suggesting that overexpression of M68 may precede amplification in tumors. Finally, we find that M68 lies within a four-gene cluster that includes a
n
ovel
h
elicase-
l
ike gene (NHL) related to RAD3/ERCC2, a plasma membrane Ras-related GTPase and a member of the stathmin family, amplification or overexpression of which may also contribute to cell growth and tumor progression.
Arthropods: Developmental diversity within a (super) phylum
Authors: Akam Michael
Published: July 2002

The expression patterns of developmental genes provide new markers
that address the homology of body parts and provide clues as to how
body plans have evolved. Such markers support the idea that insect
wings evolved from limbs but refute the idea that insect and crustacean
jaws are fundamentally different in structure. They also confirm that
arthropod tagmosis reflects underlying patterns of Hox gene regulation
but they do not yet resolve to what extent Hox expression domains may
serve to define segment homologies.
Vertebrate innovations
Authors: Shimeld Sebastian M. , Holland Peter W. H.
Published: July 2002

Vertebrate innovations include neural crest cells and their
derivatives, neurogenic placodes, an elaborate segmented brain,
endoskeleton, and an increase in the number of genes in the genome.
Comparative molecular and developmental data give new insights into the
evolutionary origins of these characteristics and the complexity of the
vertebrate body.
Need for cognitive closure and coping strategies
Authors: Kosic Ankica
Published: July 2002

This study investigated the hypothesis that the process of coping may be motivated by an interaction of directional motivational factors represented by job satisfaction/dissatisfaction and by non‐directional or epistemological motivational factors represented by the level of Need for Cognitive Closure. Need for Cognitive Closure is based on two general tendencies: the urgency tendency (“to seize”) and the permanency tendency (“to freeze”). The urgency tendency reflects the desire to attain closure as quickly as possible (“to seize”) whereas the permanence tendency refers to the inclination to maintain closure for as long as possible along with a desire to preserve or “freeze on” past knowledge and to safeguard future knowledge. The urgency and permanence notions both rest on the assumption that people under a heightened need for closure experience its absence as aversive. In relation with this, we predicted that if job satisfaction is low, the increased need for closure is related to the choice of problem‐oriented coping strategies. Alternatively, we hypothesized that with high job satisfaction an increased need for closure is related to use of avoidance coping. Questionnaires pertaining to need for cognitive closure, to coping strategies and to a measure of job satisfaction were completed by a group of 146 Croatian immigrants living in Italy. Results of the analyses confirmed that when subjects were highly satisfied with their job, their primary concern was to preserve their position. So, here the high need for closure enhanced the tendency to freeze and induced the choice of avoidance coping strategies. On the other hand, our results confirmed that when persons are not satisfied with their job, high need for closure increases the desire for change and improvement. Immigrants increased the tendency to seize manifests itself in extensive and quickened information processing relative to the use of problem‐oriented coping strategies.
Northern Ireland: Democratizing for Development
Authors: Morrissey Mike , Gaffikin Frank
Published: July 2002

This introductory article does three things. First, it indicates the general purpose of this special edition and outlines the structure and intention of the four main articles that follow. Secondly, it elaborates the arguments and theoretical discourse underpinning the articles, and thirdly, it locates these concerns and development initiatives not only in the specific regional context of Northern Ireland, but also in the wider framework of a new global economy.
Partnerships and Policy in Northern Ireland
Authors: Murtagh Brendan
Published: July 2002

This paper evaluates the experiences of the Derry/Lon don derry Partnership Board established as part of the European Union's (EU) Peace and Reconciliation Programme in Northern Ireland and suggests that it has been able to establish a distinctive approach to community consultation , strategy formulation and programme delivery. It explores the method and content of its most recent Action Plan and argues that the fealty that has traditionally informed local government discourses has been challenged by an open , pluralist and respectful analysis of position s an d problems. The paper concludes by arguing that the real contribution of the Partnerships have been to create the space for civic democracy to be played out in an attempt to embed the peace process and a long-term political settlement.
Dematerializing Local Economies: A Case for ad hoc Governance
Authors: Webber Don J. , Gore Tony
Published: July 2002

Part of the decentring process that has evolved with the relative empowerment of sub-national actors has permitted the growth of policy networks. This paper explores which type of governance structure is required to affect unemployment and growth rates when the economy comprises workers in dematerialized firms. Governance is found to influence the regional success of a ‘weightless economy’, but not in a manner that necessarily requires precise strata. Instead, as dematerialized firms are composed of potentially highly mobile workers, policies should be geared towards attracting (and keeping) mobile workers (not companies) and hence governance needs to be flexible and ad hoc to meet the locational tastes of such workers. Benefits will then spill over into the locale through increased aggregate demand for tangible goods, thereby reducing local unemployment and stimulating local growth.
Barriers to Involvement: the Disconnected Worlds of Disability and Regeneration
Authors: Edwards Claire
Published: July 2002

Disabled people are noticeably absent from the government's regeneration agenda, despite its current emphasis on empowering and involving local communities in urban renewal. This paper explores some of the barriers to disabled people's involvement in regeneration initiatives at the local level, focusing particularly on the Single Regeneration Budget (SRB). Interviews with regeneration managers, local authority officers, disabled people and disability groups identified a range of barriers. These included a lack of strategic recognition that disabled people were ‘relevant’ to regeneration, difficulties with the SRB's centrally-prescribed outputs and timescales, a lack of accessible information for disabled people, and circumscribed local political networks which served to marginalize certain disability groups from local regeneration processes. The paper concludes by suggesting ways in which some of these barriers might be addessed.
Utilization of Psychosocial Treatments by Patients Diagnosed with Bipolar Disorder and Substance Dependence
Authors: Weiss Roger D. , Kolodziej Monika E. , Najavits Lisa M. , Greenfield Shelly F. , Fucito Lisa M.
Published: July 2002

We investigated psychosocial treatment interventions, mood symptoms, and substance use among 24 patients with bipolar disorder and substance dependence. Patients were assessed for 6 months following hospital discharge. Psychotherapy and Alcoholics Anonymous (AA) attendance decreased over time. Moreover, the focus of patients' psychotherapy changed over time, with decreasing emphasis on the patients' specific disorders. Mood symptoms and substance use did not change significantly over time, although there was a trend toward more frequent drug use over time. These findings point to infrequent utilization over time of psychosocial treatments focusing specifically on bipolar and substance use disorder.
Perinatal Lead Exposure Alters the Expression of Neuronal Nitric Oxide Synthase in Rat Brain
Authors: Chetty C. S. , Reddy G. R. , Murthy K. S. , Johnson J. , Sajwan K. , Desaiah D.
Published: July 2002

Environmental exposure to lead (Pb) is known to affect the developing nervous system causing cognitive deficits in children. The diffusible nitric oxide (NO) is a biological messenger known to be involved in brain development. We examined the developmental changes of neuronal nitric oxide synthase (nNOS) in cerebellum and hippocampus of developing rat brain by radiometric assay, Western blot analysis and immunohistochemistry. Pb-exposure (0.2% Pb acetate) was initiated on gestation day 6 through the drinking water of the dam and continued through birth and postnatal days (PNDs) 1 to 21. The pups were never exposed to Pb directly. Pb exposure was stopped on weaning of pups from mothers on PND 21. The changes in nNOS were measured in the offspring on PNDs 7, 14, 21, and 35. The nNOS activity was increased gradually from PNDs 7 to 35 in both cerebellum and hippocampus of control rats when the enzyme activity was determined in the presence of either 0.5 or 6 μM calcium (Ca2+) in the reaction mixture. However, Pb exposure decreased the nNOS activity significantly at PNDs 21 to 35 as compared to respective controls when the enzyme activity was determined in the presence of 6 μM Ca2+. The decrease of nNOS was even greater and evident at all PNDs tested when the enzyme activity was assayed in the presence of physiological concentration of Ca2+ (0.5 μM). These findings were further strengthened by the in vitro studies. The cerebellar nNOS activity was inhibited much more at low Ca2+ (0.5 μM) as compared to 6 μM Ca2+, with IC50 values of 35 and 50 nM Pb, respectively. The nNOS protein levels and immunoreactivity in the cerebellum and hippocampus of rats perinatally exposed to Pb were decreased as compared to controls at PNDs 21 and 35. These data suggest perinatal Pb exposure decreases the nNOS in the developing brain. The decrease of nNOS activity and protein may explain the Pb-mediated cognitive deficits because NO regulates long-term potentiation (LTP) and other neurophysiological events in the developing nervous system.
Toxicity Evaluation of Petroleum Blending Streams: Inhalation Subchronic Toxicity/Neurotoxicity Study of a Light Catalytic Cracked Naphtha Distillate in Rats
Authors: Lapin C. , Bui Q. , Breglia R. , Koschier F. , Podhasky P. , Lapadula E. , Roth R. , Schreiner C. , White R. , Clark C. , Mandella R. , Hoffman G.
Published: July 2002

A 15-week, whole-body inhalation study of the vapors of a distillate (LCCN-D) of light catalytic cracked naphtha (CAS no. 64741–55–5, LCCN) was conducted with Sprague-Dawley rats. Target exposure concentrations were 0, 750, 2500, and 7500 ppm for 6 hours/day, 5 days/week. Over the course of the study, animals received at least 65 exposures. For a portion of the control and 7500-ppm groups, a 4-week postexposure period was included in the study. Subchronic toxicity was evaluated using standard parameters. During life, neurotoxicity was evaluated by motor activity assessment and a functional observational battery. Selected tissues from animals in all exposure groups were examined microscopically. Neuropathologic examination of selected neuronal tissues from animals in the control and high-exposure groups was also conducted. No compound-related effects were seen on survival, clinical chemistry, food consumption, or physical signs. No evidence of neurotoxicity was seen at any exposure level. Slight decreases in hematocrit and hemoglobin concentrations were seen in male rats at the end of exposure to 7500 ppm LCCN-D. However, values were within normal physiological ranges and recovery occurred. Slight decreases in mean body weights and body weight gain were observed in high-exposure females during the first 7 weeks of exposure, but this decrease was not seen during the second half of the study. Male rat nephropathy involving hyaline droplet formation and alpha-2μ-globulin accumulation was seen in mid-and high-exposure males, an effect not relevant to humans. The incidence and severity of goblet cell hypertrophy/hyperplasia and respiratory epithelium hyperplasia in nasoturbinal tissues were greater in high-exposure animals, but recovery occurred. None of the effects observed were considered toxicologically significant. The no-observable-adverse-effect level (NOAEL) for subchronic and neurotoxicity of LCCN-D was ≥7500 ppm.
The Blockade of Mineralocorticoid Hormone Signaling Provokes Dramatic Teratogenesis in Cultured Rat Embryos
Authors: Mirshahi M. , Ayani E. , Nicolas C. , Golestaneh N. , Ferrari P. , Valamanesh F. , Agarwal M. K.
Published: July 2002

Although the administration of adrenocortical hormones to pregnant rats provokes only limited effect on the growth and development of the fetus, the direct influence of these steroids on cultured embryos has never been studied. The disruption of cell signaling by ZK 91587, which specifically occupies the mineralocorticoid receptor, resulted within 2 days in significant and pronounced adverse effects on the total length, the somite number, the embryo curvature, the communication between vitelline and ombilical blood vessels in the allantoid, and the vascularization of the vitelline sac, in 244-hour Wistar rat embryos in culture. The average score of 16 organs declined in a dose-dependant manner, following exposure to ZK 91587, and this was totally reversed by 10 μM aldosterone which, by itself, did not at all influence the embryonic development. The organogenesis was inhibited in the order: hind limb > fore limb > optic stalk > brain > olfactory pit > otic vesicle. ZK 91587 was completely ineffective in embryos that had attained the age of 260 hours. Similar, but less dramatic, results were obtained with the mineralocorticoid antagonist RU 26752, and with the antiglu-cocorticoid RU 38486. Sprague-Dawley rat embryos responded in a manner similar to the Wistar conceptuses. Thus, steroid receptor-mediated cell signaling is of critical importance to the growth and development of cultured rat embryos, which form a new model system to unravel adrenocortical hormone action.
Nonclinical Safety Assessment of Intravenous Optison®: A Perfluoropropane (PFP)-Filled Albumin Microspheres Contrast Agent for Ultrasonography
Authors: Greener Yigal , Killam Anne L. , Cornell Stephen T. , Osheroff Merrill R. , Wolford Suzanne T.
Published: July 2002

OPTISON (FS069), an ultrasonic diagnostic contrast agent, is a suspension of perfluoropropane (PFP)-filled album in microspheres of 2.0-4.5 microns average diameter and 5.0-8.O x 108 microspheresl ml. The following non clinical safety assessment studies in support of product regulatory submissions were conducted: genetic toxicology, single dose (rat, dog, monkey) and repeated (rat, dog) dose toxicology studies, hemodynamics, rabbit irritation, and in vitro blood compatibility. Dosages used throughout the studies were: 0.25, 5.0, 10, 20, or 25 ml/kg. OPTISON was nongenotoxic and nonirritant, and was compatible with human blood. It did not elicit adverse effects in single-dose studies at dosages of up to (and including) 20 ml/ kg. Initial studies conducted in rats and dogs with OPTISON repeated administration, 3 times per week for 3 weeks, showed no adverse effects in dogs to 20 ml/ kg and rats to 5 ml/kg. Mortality and adverse effects were noted in rats at dosages of 20 and 10 ml/ kg and were associated with acute pulmonary congestion. Subsequent repeated administrations for 29 and 31 consecutive days in rats and dogs, respectively, at 10 ml/kg, were not associated with adverse events, except for those which resulted from the anticipated immunogenic response to the IV administration of OPTISON human albumin-based microspheres. No changes in hemodynamic parameters attributable to OPTISON were reported at a dosage of 0.25 ml/ kg in dogs. It is, therefore, concluded that the intravenous administration of OPTISON, at dosages and dose regimens used in these studies, provides a large safety margin for the clinical efficacious dose for diagnostic ultrasound imaging.
Alcohol and the pancreas
Authors: APTE MINOTI V. , HABER PAUL S. , NORTON IAN D. , WILSON JEREMY S.
Published: July 2002

Alcoholic pancreatitis is a major, often lethal complication of alcohol abuse. Until recently it was generally accepted that alcoholic pancreatitis was a chronic disease from the outset. However, there is now emerging evidence in favour of the necrosis‐fibrosis hypothesis that alcoholic pancreatitis begins as an acute process and that repeated acute attacks lead to chronic pancreatitis, resulting in exocrine and endocrine failure. Over the past 10‐15 years, the focus of research into the pathogenesis of alcoholic pancreatitis has shifted from possible sphincteric and ductular abnormalities to the acinar cell itself which has increasingly been implicated as the initial site of injury. Recent studies have shown that the acinar cell can metabolize alcohol at rates comparable to those observed in hepatocytes. In addition, it has been demonstrated that alcohol and its metabolites exert direct effects on the pancreatic acinar cell which may promote premature digestive enzyme activation and oxidant stress. The challenge remains to identify predisposing and triggering factors in this disease.
Epidermal Growth Factor Receptor Signal Transactivation
Authors: Leserer Michael , Gschwind Andreas , Ullrich Axel
Published: July 2002

Cross‐communication between heterologous signaling systems and the epidermal growth factor receptor (EGFR) has been shown to be critical for a variety of biological responses: EGFR transactivation when G‐protein‐coupled receptors (GPCRs) are stimulated represents the paradigm of an interreceptor network that is dependent on G‐proteins, kinases, metalloproteases, and growth factor precursors. Investigating the mechanism of this process will help expand our knowledge of physiological regulatory mechanisms and diverse pathophysiological disorders.
Caveolin-3 Associates with Developing T-tubules during Muscle Differentiation
Authors: Parton Robert G. , Way Michael , Zorzi Natasha , Stang Espen
Published: July 2002

Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells. We have recently cloned a muscle-specific caveolin, termed caveolin-3, which is expressed in differentiated muscle cells. Specific antibodies to caveolin-3 were generated and used to characterize the distribution of caveolin-3 in adult and differentiating muscle. In fully differentiated skeletal muscle, caveolin-3 was shown to be associated exclusively with sarcolemmal caveolae. Localization of caveolin-3 during differentiation of primary cultured muscle cells and development of mouse skeletal muscle in vivo suggested that caveolin-3 is transiently associated with an internal membrane system. These elements were identified as developing transverse-(T)-tubules by double-labeling with antibodies to the α1 subunit of the dihydropyridine receptor in C2C12 cells. Ultrastructural analysis of the caveolin-3– labeled elements showed an association of caveolin-3 with elaborate networks of interconnected caveolae, which penetrated the depths of the muscle fibers. These elements, which formed regular reticular structures, were shown to be surface-connected by labeling with cholera toxin conjugates. The results suggest that caveolin-3 transiently associates with T-tubules during development and may be involved in the early development of the T-tubule system in muscle.
Carcinoembryonic Antigen, a Human Tumor Marker, Cooperates with Myc and Bcl-2 in Cellular Transformation
Authors: Screaton Robert A. , Penn Linda Z. , Stanners Clifford P.
Published: July 2002

Carcinoembryonic antigen (CEA) is a tumor marker that is overexpressed in many human cancers and functions in vitro as a homotypic intercellular adhesion molecule. We have investigated the possibility of synergy between CEA, v-Myc, and Bcl-2 in the transformation of cells with differentiation capacity. We find that v-Myc increases the cell division rate and maximum density of rat L6 myoblasts but also markedly stimulates both apoptosis and surprisingly, differentiation, thus preventing transformation. The superposition of Bcl-2 blocks the apoptotic stimulation of v-Myc and independently promotes further cell division at confluence, but still allows differentiation. The further expression of CEA has a dominant effect in blocking differentiation, regardless of the presence of the other activated oncogenes, generating cells that enter a reversible quiescent G0-like state in medium promoting differentiation. Transfectants expressing CEA with or without v-myc and bcl-2 allow the emergence of cells with the property of heritable, efficient, anchorageindependent growth in soft agar and the ability to markedly reduce the latency for tumor formation in nude mice. We propose that by prolonging cell survival in the presence of differentiation signals, CEA represents a novel class of dominant differentiation-blocking oncogene.
Glucose Transporter (GLUT-4) Is Targeted to Secretory Granules in Rat Atrial Cardiomyocytes
Authors: Slot Jan W. , Garruti Gabriella , Martin Sally , Oorschot Viola , Posthuma George , Kraegen Edward W. , Laybutt Ross , Thibault Gaétan , James David E.
Published: July 2002

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4–containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.
Heterotrimeric Kinesin-II Is Required for the Assembly of Motile 9+2 Ciliary Axonemes on Sea Urchin Embryos
Authors: Morris Robert L. , Scholey Jonathan M.
Published: July 2002

Heterotrimeric kinesin-II is a plus end– directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fertilized sea urchin eggs with an anti–kinesin-II monoclonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of short, paralyzed, 9+0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9+2 cilia that contain kinesin-II as detected by Western blotting. Injection of anti–kinesin-II into one blastomere of a two-cell embryo leads to the development of chimeric blastulae covered on one side with short, paralyzed cilia, and on the other with normal, beating cilia. We observed a unimodal length distribution of short cilia on anti–kinesin-II–injected embryos corresponding to the first mode of the trimodal distribution of ciliary lengths observed for control embryos. This short mode may represent a default ciliary assembly intermediate. We hypothesize that kinesin-II functions during ciliogenesis to deliver ciliary components that are required for elongation of the assembly intermediate and for formation of stable central pair MTs. Thus, kinesin-II plays a critical role in embryonic development by supporting the maturation of nascent cilia to generate long motile organelles capable of producing the propulsive forces required for swimming and feeding.
A Dominant-negative Clathrin Mutant Differentially Affects Trafficking of Molecules with Distinct Sorting Motifs in the Class II Major Histocompatibility Complex (MHC) Pathway
Authors: Liu Shu-Hui , Marks Michael S. , Brodsky Frances M.
Published: July 2002

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3γ, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA–DM and the invariant chain (I chain) complexed to HLA–DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR–I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR–I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR–I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR–I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR–I chain until their intended rendezvous in late endocytic compartments.
A New Member of the Rho Family, Rnd1, Promotes Disassembly of Actin Filament Structures and Loss of Cell Adhesion
Authors: Nobes Catherine D. , Lauritzen Inger , Mattei Marie-Geneviève , Paris Sonia , Hall Alan , Chardin Pierre
Published: July 2002

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.
Functional Specialization of Stable and Dynamic Microtubules in Protein Traffic in WIF-B Cells
Authors: Poüs C. , Chabin K. , Drechou A. , Barbot L. , Phung-Koskas T. , Settegrana C. , Bourguet-Kondracki M.L. , Maurice M. , Cassio D. , Guyot M. , Durand G.
Published: July 2002

We found that the magnesium salt of ilimaquinone, named 201-F, specifically disassembled dynamically unstable microtubules in fibroblasts and various epithelial cell lines. Unlike classical tubulin- interacting drugs such as nocodazole or colchicine which affect all classes of microtubules, 201-F did not depolymerize stable microtubules. In WIF-B–polarized hepatic cells, 201-F disrupted the Golgi complex and inhibited albumin and alpha1-antitrypsin secretion to the same extent as nocodazole. By contrast, 201-F did not impair the transport of membrane proteins to the basolateral surface, which was only affected by the total disassembly of cellular microtubules. Transcytosis of two apical membrane proteins—the alkaline phosphodiesterase B10 and dipeptidyl peptidase IV—was affected to the same extent by 201-F and nocodazole. Taken together, these results indicate that only dynamically unstable microtubules are involved in the transport of secretory proteins to the plasma membrane, and in the transcytosis of membrane proteins to the apical surface. By contrast, stable microtubules, which are not functionally affected by 201-F treatment, are involved in the transport of membrane proteins to the basolateral surface. By specifically disassembling highly dynamic microtubules, 201-F is an invaluable tool with which to study the functional specialization of stable and dynamic microtubules in living cells.
UNC-60B, an ADF/Cofilin Family Protein, Is Required for Proper Assembly of Actin into Myofibrils in Caenorhabditis elegans Body Wall Muscle
Authors: Ono Shoichiro , Baillie David L. , Benian Guy M.
Published: July 2002

The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils.