bluuebunny/crossref_metadata_2025 huggingface dataset:
LARD: A new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing
Authors: Screaton Gavin R. , Xu Xiao-Ning , Olsen Anna L. , Cowper Alison E. , Tan Rusung , McMichael Andrew J. , Bell John I.
Published: July 2002

Fas and TNF-R1 are cysteine-rich cell surface receptors related to the low-affinity nerve growth factor receptor family. Engagement of these receptors by their respective ligands, FasL and tumor necrosis factor, leads to apoptosis that is signaled through a conserved intracellular portion of the receptor termed the “death domain.” We have cloned a new member of this family, lymphocyte-associated receptor of death (LARD), which leads to spontaneous apoptosis when expressed in 293T cells. The expression of LARD is more tightly regulated than that of either Fas or TNF-R1 as it is found predominantly on lymphocytes (T and B cells) but not on macrophages or a number of transformed lymphocyte cell lines. Alternative pre-mRNA splicing generates at least 11 distinct isoforms of LARD. The full-length isoform, LARD-1, extends to include the transmembrane and death domains, whereas the other isoforms encode potentially secreted molecules. Naive B and T cells express very little LARD-1 but express combinations of the other isoforms. Upon T cell activation, a programmed change in alternative splicing occurs so that the full-length, membrane-bound LARD-1 predominates. This may have implications for the control of lymphocyte proliferation following activation.
Wnt signaling and transcriptional control of
Siamois
in
Xenopus
embryos
Authors: Fan Melinda J. , Grüning Wolfram , Walz Gerd , Sokol Sergei Y.
Published: July 2002

The Wnt-inducible homeobox gene
Siamois
is expressed in
Xenopus
embryos before gastrulation and is necessary for formation of the Spemann organizer. Here we show that 5′-flanking sequences of the
Siamois
coding region can specifically activate a heterologous reporter gene in dorsovegetal cells, thus mimicking
Siamois’s
endogenous expression. A 245-bp DNA fragment is sufficient for activation by both Wnts and endogenous inducers. A dominant negative form of
Xenopus
T cell-specific factor 3 (XTCF-3) inhibited promoter activity, indicating that T cell-specific factor (TCF)/lymphocyte enhancer binding factor 1 (LEF-1) signaling is necessary for regulation of
Siamois
. Mutagenesis of two individual TCF sites in the −245 promoter revealed that the proximal, but not distal, site is necessary for dorsovegetal activation. These observations suggest that
Siamois
is directly regulated by TCFs during dorsoventral axis determination. Further deletion analysis identified a positive regulatory region that is required for dorsal activation, but not for Wnt inducibility, of the promoter. We also present evidence for autoregulation of
Siamois
transcription. Furthermore, the
Siamois
promoter was activated by Wnt signaling in 293T tissue culture cells, demonstrating that regulation of the promoter is functionally conserved.
The mouse and human genes encoding the recognition component of the N-end rule pathway
Authors: Kwon Yong Tae , Reiss Yuval , Fried Victor A. , Hershko Avram , Yoon Jeong Kyo , Gonda David K. , Sangan Pitchai , Copeland Neal G. , Jenkins Nancy A. , Varshavsky Alexander
Published: July 2002

The N-end rule relates the
in vivo
half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human
Ubr1
cDNAs and genes that encode a mammalian N-recognin called E3α. Mouse UBR1p (E3α) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast
Saccharomyces cerevisiae
. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain.
Ubr1
is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse
Ubr1
spans ≈120 kilobases of genomic DNA and contains ≈50 exons.
Ubr1
is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the
Ubr1
expression is highest in the branchial arches and in the tail and limb buds. The cloning of
Ubr1
makes possible the construction of
Ubr1
-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.
Inhibition of Stat1-mediated gene activation by PIAS1
Authors: Liu Bin , Liao Jiayu , Rao Xiaoping , Kushner Steven A. , Chung Chan D. , Chang David D. , Shuai Ke
Published: July 2002

STAT (signal transducer and activator of transcription) proteins are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation in response to cytokine stimulation. Tyrosine phosphorylated STATs dimerize and translocate into the nucleus to activate specific genes. Different members of the STAT protein family have distinct functions in cytokine signaling. Biochemical and genetic analysis has demonstrated that Stat1 is essential for gene activation in response to interferon stimulation. Although progress has been made toward understanding STAT activation, little is known about how STAT signals are down-regulated. We report here the isolation of a family of PIAS (protein inhibitor of activated STAT) proteins. PIAS1, but not other PIAS proteins, blocked the DNA binding activity of Stat1 and inhibited Stat1-mediated gene activation in response to interferon. Coimmunoprecipitation analysis showed that PIAS1 was associated with Stat1 but not Stat2 or Stat3 after ligand stimulation. The
in vivo
PIAS1–Stat1 interaction requires phosphorylation of Stat1 on Tyr-701. These results identify PIAS1 as a specific inhibitor of Stat1-mediated gene activation and suggest that there may exist a specific PIAS inhibitor in every STAT signaling pathway.
Uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis
Authors: Hooper D. C. , Spitsin S. , Kean R. B. , Champion J. M. , Dickson G. M. , Chaudhry I. , Koprowski H.
Published: July 2002

Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-γ receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.
Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function
Authors: Barton-Davis Elisabeth R. , Shoturma Daria I. , Musaro Antonio , Rosenthal Nadia , Sweeney H. Lee
Published: July 2002

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.
The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D
Authors: Cocchi Francesca , Lopez Marc , Menotti Laura , Aoubala Mustapha , Dubreuil Patrice , Campadelli-Fiume Gabriella
Published: July 2002

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1.302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for
in vitro
physical interaction with gD. The
in vitro
binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.
Attenuation of the Langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4
Authors: Pletnev Alexander G. , Men Ruhe
Published: July 2002

Langat virus (LGT) strain TP21 is the most attenuated of the tick-borne flaviviruses for humans. Even though LGT has low-level neurovirulence for humans, it, and its more attenuated egg-passage derivative, strain E5, exhibit significant neurovirulence and neuroinvasiveness in normal mice, albeit less than that associated with tick-borne encephalitis virus (TBEV), the most virulent of the tick-borne flaviviruses. We sought to reduce or ablate these viral phenotypes of TP21 and E5 by using a strategy that had been used successfully in the past to reduce neurovirulence and abolish neuroinvasiveness of TBEV, namely substitution of structural protein genes of the tick-borne flavivirus for the corresponding genes of dengue type 4 virus (DEN4). In pursuit of these objectives different combinations of LGT genes were substituted into the DEN4 genome but only chimeras containing LGT structural proteins premembrane (preM) and envelope glycoprotein (E) were viable. The infectious LGT(preM-E)/DEN4 chimeras were restricted in replication in simian cell cultures but grew to moderately high titer in mosquito cell culture. Also, the chimeras were at least 5,000 times less neurovirulent than their parental LGT virus in suckling mice. Significantly, the chimeras lacked detectable evidence of neuroinvasiveness after i.p. inoculation of Swiss mice or the more permissive SCID mice with 105or 107plaque-forming units (PFU), respectively. Nonetheless, i.p. inoculation of Swiss mice with 10 or 103PFU of either chimeric virus induced LGT neutralizing antibodies and resistance to fatal encephalitis caused by i.p. challenge with LGT TP21. The implications of these observations for development of a live attenuated TBEV vaccine are discussed.
Skeletal overgrowth in transgenic mice that overexpress brain natriuretic peptide
Authors: Suda Michio , Ogawa Yoshihiro , Tanaka Kiyoshi , Tamura Naohisa , Yasoda Akihiro , Takigawa Toshiya , Uehira Masahiro , Nishimoto Hirofumi , Itoh Hiroshi , Saito Yoshihiko , Shiota Kohei , Nakao Kazuwa
Published: July 2002

Longitudinal bone growth is determined by the process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones and involves many systemic hormones and local regulators. Natriuretic peptides organize a family of three structurally related peptides: atrial natriuretic peptide, brain natriuretic peptide (BNP), and C-type natriuretic peptide. Atrial natriuretic peptide and BNP are cardiac hormones that are produced predominantly by the atrium and ventricle, respectively. C-type natriuretic peptide occurs in a wide variety of tissues, where it acts as a local regulator. These peptides can influence body fluid homeostasis and blood pressure control through the activation of two guanylyl cyclase (GC)-coupled natriuretic peptide receptor subtypes—GC-A and GC-B. We report here marked skeletal overgrowth in transgenic mice that overexpress BNP. Transgenic mice with elevated plasma BNP concentrations exhibited deformed bony skeletons characterized by kyphosis, elongated limbs and paws, and crooked tails. Bone abnormalities resulted from a high turnover of endochondral ossification accompanied by overgrowth of the growth plate. Studies using an
in vitro
organ culture of embryonic mouse tibias revealed that BNP increases the height of cartilaginous primordium directly, thereby stimulating the total longitudinal bone growth. The present study demonstrates that natriuretic peptides can affect the process of endochondral ossification.
The role of inducible nitric oxide synthase in the host response to Coxsackievirus myocarditis
Authors: Zaragoza Carlos , Ocampo Christopher , Saura Marta , Leppo Michelle , Wei Xiao-Qing , Quick Richard , Moncada Salvador , Liew Foo Y. , Lowenstein Charles J.
Published: July 2002

The host response to Coxsackievirus infection is complex, including T lymphocytes, B lymphocytes, natural killer cells, and macrophages. Although Coxsackievirus infection induces expression of inducible nitric oxide synthase (NOS2; EC1.14.13.39) in macrophages, the precise role of NOS2 in the host response to Coxsackievirus myocarditis has been unclear. We show, by using mice homozygous for a disrupted NOS2 allele, that Coxsackievirus replicates to higher titers in NOS2−/−mice, that the host lacking NOS2 clears virus more slowly than the wild-type host, and that myocarditis is much more severe in infected NOS2−/−mice. These data show that NOS2 is crucial for the host response to Coxsackievirus in the mouse.
Neural correlates of the episodic encoding of pictures and words
Authors: Grady Cheryl L. , McIntosh Anthony R. , Rajah M. Natasha , Craik Fergus I. M.
Published: July 2002

A striking characteristic of human memory is that pictures are remembered better than words. We examined the neural correlates of memory for pictures and words in the context of episodic memory encoding to determine material-specific differences in brain activity patterns. To do this, we used positron emission tomography to map the brain regions active during encoding of words and pictures of objects. Encoding was carried out by using three different strategies to explore possible interactions between material specificity and types of processing. Encoding of pictures resulted in greater activity of bilateral visual and medial temporal cortices, compared with encoding words, whereas encoding of words was associated with increased activity in prefrontal and temporoparietal regions related to language function. Each encoding strategy was characterized by a distinctive activity pattern, but these patterns were largely the same for pictures and words. Thus, superior overall memory for pictures may be mediated by more effective and automatic engagement of areas important for visual memory, including medial temporal cortex, whereas the mechanisms underlying specific encoding strategies appear to operate similarly on pictures and words.
Stimulation of renin secretion by nitric oxide is mediated by phosphodiesterase 3
Authors: Kurtz Armin , Götz Karl-Heinz , Hamann Marlies , Wagner Charlotte
Published: July 2002

This study aimed to characterize the cellular pathways along which nitric oxide (NO) stimulates renin secretion from the kidney. Using the isolated perfused rat kidney model we found that renin secretion stimulated 4- to 8-fold by low perfusion pressure (40 mmHg), by macula densa inhibition (100 μmol/liter of bumetanide), and by adenylate cyclase activation (3 nmol/liter of isoproterenol) was markedly attenuated by the NO synthase inhibitor nitro-
l
-arginine methyl ester (
l
-Name) (1 mM) and that the inhibition by
l
-Name was compensated by the NO-donor sodium nitroprusside (SNP) (10 μmol/liter). Similarly, inhibition of cAMP degradation by blockade of phosphodiesterase 1 (PDE-1) (20 μmol/liter of 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine) or of PDE-4 (20 μmol/liter of rolipram) caused a 3- to 4-fold stimulation of renin secretion that was attenuated by
l
-Name and that was even overcompensated by sodium nitroprusside. Inhibition of PDE-3 by 20 μmol/liter of milrinone or by 200 nmol/liter of trequinsin caused a 5- to 6-fold stimulation of renin secretion that was slightly enhanced by NO synthase inhibition and moderately attenuated by NO donation. Because PDE-3 is a cGMP-inhibited cAMP-PDE the role of endogenous cGMP for the effects of NO was examined by the use of the specific guanylate cyclase inhibitor 1-H-(1,2,4)oxodiazolo(4,3a)quinoxalin-1-one (20 μmol). In the presence of 1
H
-[1,2,4]oxodiazolo[4,3-
a
]quinoxalin-1-one the effect of NO on renin secretion was abolished, whereas PDE-3 inhibitors exerted their normal effects. These findings suggest that PDE-3 plays a major role for the cAMP control of renin secretion. Our findings are compatible with the idea that the stimulatory effects of endogenous and exogenous NO on renin secretion are mediated by a cGMP-induced inhibition of cAMP degradation.
Selective, covalent modification of β-tubulin residue Cys-239 by T138067, an antitumor agent with
in vivo
efficacy against multidrug-resistant tumors
Authors: Shan Bei , Medina Julio C. , Santha Edit , Frankmoelle Walter P. , Chou Ting-C. , Learned Robert M. , Narbut Mathew R. , Stott Dean , Wu Pengguang , Jaen Juan C. , Rosen Terry , Timmermans Pieter B. M. W. M. , Beckmann Holger
Published: July 2002

Microtubules are linear polymers of α- and β-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis. Here we describe a synthetic compound, 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the β
1
, β
2
, and β
4
isotypes of β-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymerization. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clinically useful for the treatment of multidrug-resistant tumors.
The γ-subunit rotation and torque generation in F
1
-ATPase from wild-type or uncoupled mutant
Escherichia coli
Authors: Omote Hiroshi , Sambonmatsu Noriko , Saito Kiwamu , Sambongi Yoshihiro , Iwamoto-Kihara Atsuko , Yanagida Toshio , Wada Yoh , Futai Masamitsu
Published: July 2002

The rotation of the γ-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase (F
O
F
1
). The
Escherichia coli
ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A fluorescently labeled actin filament connected to the γ-subunit of the F
1
sector rotated on addition of ATP. This progress enabled us to analyze the γM23K (the γ-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the F
1
sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the γM23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.
Nuclear localization of the p120
ctn
Armadillo-like catenin is counteracted by a nuclear export signal and by E-cadherin expression
Authors: van Hengel J. , Vanhoenacker P. , Staes K. , van Roy F.
Published: July 2002

The Armadillo protein p120
ctn
associates with the cytoplasmic domain of cadherins and accumulates at cell–cell junctions. Particular Armadillo proteins such as β-catenin and plakophilins show a partly nuclear location, suggesting gene-regulatory activities. For different human E-cadherin-negative carcinoma cancer cell lines we found expression of endogenous p120
ctn
in the nucleus. Expression of E-cadherin directed p120
ctn
out of the nucleus. Previously, we reported that the human p120
ctn
gene might encode up to 32 protein isoforms as products of alternative splicing. Overexpression of p120
ctn
isoforms B in various cell lines resulted in cytoplasmic immunopositivity but never in nuclear staining. In contrast, upon expression of p120
ctn
cDNAs lacking exon B, the isoforms were detectable within both nuclei and cytoplasm. A putative nuclear export signal (NES) with a characteristic leucine-rich motif is encoded by exon B. This sequence element was shown to be required for nuclear export and to function autonomously when fused to a carrier protein and microinjected into cell nuclei. Moreover, the NES function of endogenously or exogenously expressed p120
ctn
isoforms B was sensitive to the nuclear export inhibitor leptomycin B. Expression of exogenous E-cadherin down-regulated nuclear p120
ctn
whereas activation of protein kinase C increased the level of nuclear p120
ctn
. These results reveal molecular mechanisms controlling the subcellular distribution of p120
ctn
.
Induction of an acrosomal process in
Toxoplasma gondii
: Visualization of actin filaments in a protozoan parasite
Authors: Shaw Michael K. , Tilney Lewis G.
Published: July 2002

The invasive stages of
Toxoplasma gondii
, an Apicomplexan parasite, actively invade their host cells in an actin-dependent way. However, despite containing biochemically significant amounts of actin, actin filaments have never been observed in these parasites. Jasplakinolide, a membrane-permeable actin-polymerizing and filament-stabilizing drug, induced the polymerization of actin filaments at the anterior end of each tachyzoite in association with the conoid, where they formed, in many cases, a prominent membrane-enclosed apical projection reminiscent of acrosomal processes of invertebrate sperm. These jasplakinolide-induced filaments decorated with myosin subfragment 1, demonstrating unequivocally that they were indeed actin. Jasplakinolide-treated tachyzoites were unable to invade host cells, but once the drug was removed the parasites were able to enter host cells. Actin polymerization at the apical end of the parasite is consistent with the role of the apical end in host-cell invasion powered by a jackhammer-like extension and retraction of the conoid complex coupled to the secretion and rearward capping of surface proteins.
Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons
Authors: Gieffers Christian , Peters Beate H. , Kramer Edgar R. , Dotti Carlos G. , Peters Jan-Michael
Published: July 2002

The anaphase-promoting complex/cyclosome (APC) is a tightly cell cycle-regulated ubiquitin-protein ligase that targets cyclin B and other destruction box-containing proteins for proteolysis at the end of mitosis and in G1. Recent work has shown that activation of the APC in mitosis depends on CDC20, whereas APC is maintained active in G1 via association with the CDC20-related protein CDH1. Here we show that the mitotic activator CDC20 is the only component of the APC ubiquitination pathway whose expression is restricted to proliferating cells, whereas the APC and CDH1 are also expressed in several mammalian tissues that predominantly contain differentiated cells, such as adult brain. Immunocytochemical analyses of cultured rat hippocampal neurons and of mouse and human brain sections indicate that the APC and CDH1 are ubiquitously expressed in the nuclei of postmitotic terminally differentiated neurons. The APC purified from brain contains all core subunits known from proliferating cells and is tightly associated with CDH1. Purified brain APC
CDH1
has a high cyclin B ubiquitination activity that depends less on the destruction box than on the activity of mitotic APC
CDC20
. On the basis of these results, we propose that the functions of APC
CDH1
are not restricted to controlling cell-cycle progression but may include the ubiquitination of yet unidentified substrates in differentiated cells.
Selective glutamate receptor antagonists can induce or prevent axonal sprouting in rat hippocampal slice cultures
Authors: McKinney R. Anne , Lüthi Andreas , Bandtlow Christine E. , Gähwiler Beat H. , Thompson Scott M.
Published: July 2002

After the transection of the Schaffer collateral pathway in hippocampal slice cultures, reactive sprouting is induced in the CA3 area, and eventually synaptic transmission between areas CA1 and CA3 is restored. Using this model, we have studied the role of ionotropic glutamate receptors in the initiation of axonal sprouting and the regeneration of functional synapses. We show that neither reactive sprouting nor functional recovery of synaptic transmission occur in the presence of the non-N-methyl-d-aspartate (NMDA) receptor antagonist 6-nitro-7-sulfamoylbenzoquinoxaline-2,3-dione (CNQX). In contrast, the NMDA receptor antagonists methyl-10,11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801) or 3-(RS)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) did not interfere with these processes. Moreover, we observed that the application of NMDA receptor antagonists induced massive axonal sprouting and an increase in the frequency of miniature excitatory postsynaptic currents in unlesioned cultures. Our results thus indicate that NMDA and non-NMDA receptors exert a differential effect on reactive sprouting and the recovery of synaptic transmission after injury in the hippocampus. Activation of non-NMDA receptors appears necessary for these processes to occur, whereas activation of NMDA receptors suppresses growth-associated protein -43 expression and axonal outgrowth.
Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells
Authors: Lane Michelle A. , Chen Anne C. , Roman Shaun D. , Derguini Fadila , Gudas Lorraine J.
Published: July 2002

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological (≈1 μM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.
Ozone inhibits guard cell K
+
channels implicated in stomatal opening
Authors: Torsethaugen Gro , Pell Eva J. , Assmann Sarah M.
Published: July 2002

Ozone (O
3
) deleteriously affects organisms ranging from humans to crop plants, yet little is understood regarding the underlying mechanisms. In plants, O
3
decreases CO
2
assimilation, but whether this could result from direct O
3
action on guard cells remained unknown. Potassium flux causes osmotically driven changes in guard cell volume that regulate apertures of associated microscopic pores through which CO
2
is supplied to the photosynthetic mesophyll tissue. We show in
Vicia faba
that O
3
inhibits (
i
) guard cell K
+
channels that mediate K
+
uptake that drives stomatal opening; (
ii
) stomatal opening in isolated epidermes; and (
iii
) stomatal opening in leaves, such that CO
2
assimilation is reduced without direct effects of O
3
on photosynthetic capacity. Direct O
3
effects on guard cells may have ecological and agronomic implications for plant productivity and for response to other environmental stressors including drought.
Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in
Escherichia
coli
Authors: Kuroda Akio , Tanaka Shoutaro , Ikeda Tsukasa , Kato Junichi , Takiguchi Noboru , Ohtake Hisao
Published: July 2002

Inorganic polyphosphate (polyP) kinase was studied for its roles in physiological responses to nutritional deprivation in
Escherichia coli
. A mutant lacking polyP kinase exhibited an extended lag phase of growth, when shifted from a rich to a minimal medium (nutritional downshift). Supplementation of amino acids to the minimal medium abolished the extended growth lag of the mutant. Levels of the stringent response factor, guanosine 5′-diphosphate 3′-diphosphate, increased in response to the nutritional downshift, but, unlike in the wild type, the levels were sustained in the mutant. These results suggested that the mutant was impaired in the induction of amino acid biosynthetic enzymes. The expression of an amino acid biosynthetic gene,
hisG
, was examined by using a transcriptional
lacZ
fusion. Although the mutant did not express the fusion in response to the nutritional downshift, Northern blot analysis revealed a significant increase of
hisG-lacZ
mRNA. Amino acids generated by intracellular protein degradation are very important for the synthesis of enzymes at the onset of starvation. In the wild type, the rate of protein degradation increased in response to the nutritional downshift whereas it did not in the mutant. Supplementation of amino acids at low concentrations to the minimal medium enabled the mutant to express the
hisG-lacZ
fusion. Thus, the impaired regulation of protein degradation results in the adaptation defect, suggesting that polyP kinase is required to stimulate protein degradation.
Soluble complexes of regulated upon activation, normal T cells expressed and secreted (RANTES) and glycosaminoglycans suppress HIV-1 infection but do not induce Ca2+signaling
Authors: Burns Jennifer M. , Lewis George K. , DeVico Anthony L.
Published: July 2002

Chemokines comprise a family of low-molecular-weight proteins that elicit a variety of biological responses including chemotaxis, intracellular Ca2+mobilization, and activation of tyrosine kinase signaling cascades. A subset of chemokines, including regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1α (MIP-1α), and MIP-1β, also suppress infection by HIV-1. All of these activities are contingent on interactions between chemokines and cognate seven-transmembrane spanning, G protein-coupled receptors. However, these activities are strongly inhibited by glycanase treatment of receptor-expressing cells, indicating an additional dependence on surface glycosaminoglycans (GAG). To further investigate this dependence, we examined whether soluble GAG could reconstitute the biological activities of RANTES on glycanase-treated cells. Complexes formed between RANTES and a number of soluble GAG failed to induce intracellular Ca2+mobilization on either glycanase-treated or untreated peripheral blood mononuclear cells and were unable to stimulate chemotaxis. In contrast, the same complexes demonstrated suppressive activity against macrophage tropic HIV-1. Complexes composed of125I-labeled RANTES demonstrated saturable binding to glycanase-treated peripheral blood mononuclear cells, and such binding could be reversed partially by an anti-CCR5 antibody. These results suggest that soluble chemokine–GAG complexes represent seven-transmembrane ligands that do not activate receptors yet suppress HIV infection. Such complexes may be considered as therapeutic formulations for the treatment of HIV-1 infection.
Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-xL
Authors: Huang David C. S. , Hahne Michael , Schroeter Michael , Frei Karl , Fontana Adriano , Villunger Andreas , Newton Kim , Tschopp Juerg , Strasser Andreas
Published: July 2002

Fas activation triggers apoptosis in many cell types. Studies with anti-Fas antibodies have produced conflicting results on Fas signaling, particularly the role of the Bcl-2 family in this process. Comparison between physiological ligand and anti-Fas antibodies revealed that only extensive Fas aggregation, by membrane bound FasL or aggregated soluble FasL consistently triggered apoptosis, whereas antibodies could act as death agonists or antagonists. Studies on Fas signaling in cell lines and primary cells from transgenic mice revealed that FADD/MORT1 and caspase-8 were required for apoptosis. In contrast, Bcl-2 or Bcl-xLdid not block FasL-induced apoptosis in lymphocytes or hepatocytes, demonstrating that signaling for cell death induced by Fas and the pathways to apoptosis regulated by the Bcl-2 family are distinct.
The identification of markers segregating with resistance to
Schistosoma mansoni
infection in the snail
Biomphalaria glabrata
Authors: Knight Matty , Miller Andre N. , Patterson Carolyn N. , Rowe Christopher G. , Michaels George , Carr Daniel , Richards Charles S. , Lewis Fred A.
Published: July 2002

Both snail and parasite genes determine the susceptibility of
the snail
Biomphalaria glabrata
to infection with the
trematode
Schistosoma mansoni
. To identify molecular
markers associated with resistance to the parasite in the snail host,
we performed genetic crosses between parasite-resistant and
-susceptible isogenic snails. Because resistance to infection in adult
snails is controlled by a single locus, DNA samples from individual
F
2
and F
1
backcross progeny, segregating for
either the resistant or susceptible phenotypes, were pooled (bulked
segregant). Genotypes for both parents were determined with 205
arbitrary decamer primers by random amplified polymorphic DNA–PCR. Of
the 205 primers, 144 were informative, and the relative allele
frequencies between the pools for these primers were determined. Two
primers, OPM-04 and OPZ-11, produced fragments in the resistant parent
of one cross that were inherited in a dominant fashion in the resistant
F
2
and backcross-bulked segregant progeny. Subsequent
typing of DNA samples of individual progeny snails showed that the
1.2-kb marker amplified by primer OPM-04 and the 1.0-kb marker produced
by primer OPZ-11 segregated in the same dominant fashion with the
resistant phenotype. Sequence analysis of the 1.2-kb marker showed that
it corresponds to a repetitive sequence in the snail genome with no
homology to existing DNA sequences in the public databases. Analysis of
the 1.0-kb marker showed that it also corresponds to a repetitive
sequence in the
B. glabrata
genome that contains an
imperfect ORF, with homology to retrovirus-related group-specific
antigens (gag) polyprotein.
Vaccination with a recombinant vaccinia virus encoding a “self” antigen induces autoimmune vitiligo and tumor cell destruction in mice: Requirement for CD4+T lymphocytes
Authors: Overwijk Willem W. , Lee David S. , Surman Deborah R. , Irvine Kari R. , Touloukian Christopher E. , Chan Chi-Chao , Carroll Miles W. , Moss Bernard , Rosenberg Steven A. , Restifo Nicholas P.
Published: July 2002

Many human and mouse tumor antigens are normal, nonmutated tissue differentiation antigens. Consequently, immunization with these “self” antigens could induce autoimmunity. When we tried to induce immune responses to five mouse melanocyte differentiation antigens, gp100, MART-1, tyrosinase, and tyrosinase-related proteins (TRP) 1 and TRP-2, we observed striking depigmentation and melanocyte destruction only in the skin of mice inoculated with a vaccinia virus encoding mouse TRP-1. These mice rejected a lethal challenge of B16 melanoma, indicating the immune response against TRP-1 could destroy both normal and malignant melanocytes. Cytotoxic T lymphocytes specific for TRP-1 could not be detected in depigmented mice, but high titers of IgG anti-TRP-1 antibodies were present. Experiments with knockout mice revealed an absolute dependence on major histocompatibility complex class II, but not major histocompatibility complex class I, for the induction of both vitiligo and tumor protection. Together, these results suggest that the deliberate induction of self-reactivity using a recombinant viral vector can lead to tumor destruction, and that in this model, CD4+T lymphocytes are an integral part of this process. Vaccine strategies targeting tissue differentiation antigens may be valuable in cancers arising from nonessential cells and organs such as melanocytes, prostate, testis, breast, and ovary.
Intravitreous transplantation of encapsulated fibroblasts secreting the human fibroblast growth factor 2 delays photoreceptor cell degeneration in Royal College of Surgeons rats
Authors: Uteza Yves , Rouillot Jean-Sébastien , Kobetz Alexandra , Marchant Dominique , Pecqueur Sèverine , Arnaud Emmanuelle , Prats Hervé , Honiger Jiri , Dufier Jean-Louis , Abitbol Marc , Neuner-Jehle Martin
Published: July 2002

We developed an experimental approach with genetically engineered and encapsulated mouse NIH 3T3 fibroblasts to delay the progressive degeneration of photoreceptor cells in dark-eyed Royal College of Surgeons rats. These xenogeneic fibroblasts can survive in 1.5-mm-long microcapsules made of the biocompatible polymer AN69 for at least 90 days under
in vitro
and
in vivo
conditions because of their stable transfection with the gene for the 18-kDa form of the human basic fibroblast growth factor (hFGF-2). Furthermore, when transferred surgically into the vitreous cavity of 21-day-old Royal College of Surgeons rats, the microencapsulated hFGF-2-secreting fibroblasts provoked a local delay of photoreceptor cell degeneration, as seen at 45 days and 90 days after transplantation. This effect was limited to 2.08 mm
2
(45 days) and 0.95 mm
2
(90 days) of the retinal surface. In both untreated eyes and control globes with encapsulated hFGF-2-deficient fibroblasts, the rescued area (of at most 0.08 mm
2
) was significantly smaller at both time points. Although, in a few ocular globes, surgical trauma induced a reorganization of the retinal cytoarchitecture, neither microcapsule rejection nor hFGF-2-mediated tumor formation were detected in any treated eyes. These findings indicate that encapsulated fibroblasts secreting hFGF-2 or perhaps other agents can be applied as potential therapeutic tools to treat retinal dystrophies.
Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain
Authors: Lee Jeannie T. , Lu Naifung , Han Yoon
Published: July 2002

Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, theXicorchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up theXic.We previously showed that theXicis contained within a 450-kb sequence that includesXist, an RNA-encoding gene required for X inactivation. To characterize theXicfurther, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We testedXicdeletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes.XistRNA coated the autosome but skipped theXicTg, raising the possibility that X chromosome domains escape inactivation by excludingXistRNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of theXist5′ sequence resulted in the loss of somatic X inactivation without abolishingXistexpression in undifferentiated cells. Thus,Xistexpression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multipleXicconstructs and insertion sites indicated that long-rangeXiceffects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.
Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes
Authors: Liu Kebin , Schoonmaker Michele M. , Levine Bruce L. , June Carl H. , Hodes Richard J. , Weng Nan-ping
Published: July 2002

Human telomerase consists of two essential components, telomerase RNA template (hTER) and telomerase reverse transcriptase (hTERT), and functions to synthesize telomere repeats that serve to protect the integrity of chromosomes and to prolong the replicative life span of cells. Telomerase activity is expressed selectively in germ-line and malignant tumor cells but not in most normal human somatic cells. As a notable exception, telomerase is expressed in human lymphocytes during development, differentiation, and activation. Recent studies have suggested that regulation of telomerase is determined by transcription of hTERT but not hTER. The highly regulated expression of telomerase in lymphocytes provides an opportunity to analyze the contribution of transcriptional regulation of hTERT and hTER. We report here an analysis of hTERT expression by Northern andin situhybridization. It was found that hTERT mRNA is expressed at detectable levels in all subsets of human lymphocytes isolated from thymus, tonsil, and peripheral blood, regardless of the status of telomerase activity. hTERT expression is regulated as a function of lineage development, differentiation, and activation. Strikingly, however, telomerase activity in these cells is not correlated strictly with the levels of hTERT and hTER transcripts. The absence of correlation between telomerase activity and hTERT mRNA could not be attributed to the presence of hTERT splice variants or to detectable inhibitors of telomerase activity. Thus, transcriptional regulation of hTERT is not sufficient to account for telomerase activity in human lymphocytes, indicating a likely role of posttranscriptional factors in the control of enzyme function.
The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions
Authors: Murakami Tsutomu , Freed Eric O.
Published: July 2002

Lentiviruses, including HIV-1, have transmembrane envelope (Env) glycoproteins with cytoplasmic tails that are quite long compared with those of other retroviruses. However, mainly because of the lack of biochemical studies performed in cell types that are targets for HIV-1 infection, no clear consensus exists regarding the function of the long lentiviral Env cytoplasmic tail in virus replication. In this report, we characterize the biological and biochemical properties of an HIV-1 mutant lacking the gp41 cytoplasmic tail. We find that the gp41 cytoplasmic tail is necessary for the efficient establishment of a productive, spreading infection in the majority of T cell lines tested, peripheral blood mononuclear cells, and monocyte-derived macrophages. Biochemical studies using a high-level, transient HIV-1 expression system based on pseudotyping with the vesicular stomatitis virus glycoprotein demonstrate that in HeLa and MT-4 cells, mutant Env incorporation into virions is reduced only 3-fold relative to wild type. In contrast, gp120 levels in virions produced from a number of other T cell lines and primary macrophages are reduced more than 10-fold by the gp41 truncation. The Env incorporation defect imposed by the cytoplasmic tail truncation is not the result of increased shedding of gp120 from virions or reduced cell-surface Env expression. These results demonstrate that in the majority of T cell lines, and in primary cell types that serve as natural targets for HIV-1 infectionin vivo, the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions.
Cockroach diuretic hormones: Characterization of a calcitonin-like peptide in insects
Authors: Furuya Kenji , Milchak Richard J. , Schegg Kathleen M. , Zhang Jinrui , Tobe Stephen S. , Coast Geoffrey M. , Schooley David A.
Published: July 2002

Insect diuretic hormones are crucial for control of water balance.
We isolated from the cockroach
Diploptera punctata
two
diuretic hormones (DH), Dippu-DH
31
and
Dippu-DH
46
, which increase cAMP production and fluid
secretion in Malpighian tubules of several insect species.
Dippu-DH
31
and -DH
46
contain 31 and 46 amino
acids, respectively. Dippu-DH
46
belongs to the
corticotropin-releasing factor (CRF)-like insect DH family, whereas
Dippu-DH
31
has little sequence similarity to the CRF-like
DH, but is similar to the calcitonin family. Dippu-DH
46
and
-DH
31
have synergistic effects in
D.
punctata
but have only additive effects in
Locusta
migratoria
. Dippu-DH
31
represents a distinct type
of insect DH with actions that differ from those of previously
identified insect peptides with diuretic activity.