bluuebunny/crossref_metadata_2025 huggingface dataset:
Quorum-sensing acts at initiation of chromosomal replication in
Escherichia coli
Authors: Withers Helen L. , Nordström Kurt
Published: July 2002

Chromosomal replication in
Escherichia coli
was studied by flow cytometry and was found to be inhibited by an extracellular factor present in conditioned media collected during late exponential and early stationary phase, i.e., via a quorum-sensing mechanism. Our results suggest that the inhibitory activity of the extracellular factor is exerted during initiation of DNA replication rather than during elongation. Furthermore, we present evidence that this interaction may occur directly at each of the replication forks. Unlike other quorum-sensing systems described so far for Gram-negative bacteria, this inhibitory activity does not require transcription or translation to be effective. Implications of quorum-sensing regulation of DNA replication are discussed.
Constitutive achlorhydria in mucolipidosis type IV
Authors: Schiffmann Raphael , Dwyer Nancy K. , Lubensky Irina A. , Tsokos Maria , Sutliff Vincent E. , Latimer John S. , Frei Karen P. , Brady Roscoe O. , Barton Norman W. , Blanchette-Mackie E. Joan , Goldin Ehud
Published: July 2002

Mucolipidosis type IV is an autosomal recessive lysosomal storage disease of unknown etiology that causes severe neurological and ophthalmological abnormalities. In an attempt to obtain insight into the nature of the metabolic abnormality in this disorder, we prospectively evaluated 15 consecutive patients, aged 2 to 23 years, over a period of 22 months. The finding of iron deficiency in some of the patients led us to the discovery that all patients but one had markedly elevated blood gastrin levels. None had vitamin B12 deficiency. Gastroscopy in three patients showed normal gross appearance of the mucosa in two patients, 4 and 7 years old, and mucosal atrophy in a 22-year-old. Parietal cells were present in normal numbers and contained large cytoplasmic inclusions that were confirmed immunohistochemically to be lysosomal in nature. Other gastric epithelial cells appeared normal. Parietal cells contained very few tubulovesicular membranes, suggesting cellular activation, whereas apical canaliculi appeared relatively nonactivated. Both subunits of the parietal cell H
+
/K
+
-ATPase were present, and both partially colocalized with f-actin at the apical membrane. We conclude that patients with mucolipidosis type IV are constitutively achlorhydric and have partially activated parietal cells. We hypothesize that the defective protein in this disease is closely associated with the final stages of parietal cell activation and is critical for a specific type of cellular vacuolar trafficking between the cytoplasm and the apical membrane domain.
A single side chain prevents
Escherichia coli
DNA polymerase I (Klenow fragment) from incorporating ribonucleotides
Authors: Astatke Mekbib , Ng Kimmie , Grindley Nigel D. F. , Joyce Catherine M.
Published: July 2002

Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate. The Klenow fragment of
E. coli
DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold. Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2′-OH of an incoming rNTP. A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 “steric gate” can be fully effective. Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides.
Overlapping roles and asymmetrical cross-regulation of the USF proteins in mice
Authors: Sirito Mario , Lin Qun , Deng Jian Min , Behringer Richard R. , Sawadogo Michèle
Published: July 2002

USF1 and USF2 are ubiquitously expressed transcription factors implicated as antagonists of the c-Myc protooncoprotein in the control of cellular proliferation. To determine the biological role of the USF proteins, mutant mice were generated by homologous recombination in embryonic stem cells. USF1-null mice were viable and fertile, with only slight behavioral abnormalities. However, these mice contained elevated levels of USF2, which may compensate for the absence of USF1. In contrast, USF2-null mice contained reduced levels of USF1 and displayed an obvious growth defect: they were 20–40% smaller at birth than their wild-type or heterozygous littermates and maintained a smaller size with proportionate features throughout postnatal development. Some of the USF-deficient mice, especially among the females, were prone to spontaneous epileptic seizures, suggesting that USF is important in normal brain function. Among the double mutants, an embryonic lethal phenotype was observed for mice that were homozygous for the
Usf2
mutation and either heterozygous or homozygous for the
Usf1
mutation, demonstrating that the USF proteins are essential in embryonic development.
Cell adhesion and the integrin-linked kinase regulate the LEF-1 and β-catenin signaling pathways
Authors: Novak Anton , Hsu Shu-Chi , Leung-Hagesteijn Chungyee , Radeva Galina , Papkoff Jackie , Montesano Roberto , Roskelley Calvin , Grosschedl Rudolf , Dedhar Shoukat
Published: July 2002

The integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase that can interact directly with the cytoplasmic domains of the β1 and β3 integrin subunits and whose kinase activity is modulated by cell–extracellular matrix interactions. Overexpression of constitutively active ILK results in loss of cell–cell adhesion, anchorage-independent growth, and tumorigenicity in nude mice. We now show that modest overexpression of ILK in intestinal epithelial cells as well as in mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of E-cadherin expression, translocation of β-catenin to the nucleus, formation of a complex between β-catenin and the high mobility group transcription factor, LEF-1, and transcriptional activation by this LEF-1/β-catenin complex. We also find that LEF-1 protein expression is rapidly modulated by cell detachment from the extracellular matrix, and that LEF-1 protein levels are constitutively up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by activated H-
ras
or v-
src
oncogenes do not result in the activation of LEF-1/β-catenin. The results demonstrate that the oncogenic properties of ILK involve activation of the LEF-1/β-catenin signaling pathway, and also suggest ILK-mediated cross-talk between cell–matrix interactions and cell–cell adhesion as well as components of the Wnt signaling pathway.
Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit
Authors: Beernink Peter T. , Endrizzi James A. , Alber Tom , Schachman H. K.
Published: July 2002

The lack of knowledge of the three-dimensional structure of the trimeric, catalytic (C) subunit of aspartate transcarbamoylase (ATCase) has impeded understanding of the allosteric regulation of this enzyme and left unresolved the mechanism by which the active, unregulated C trimers are inactivated on incorporation into the unliganded (taut or T state) holoenzyme. Surprisingly, the isolated C trimer, based on the 1.9-Å crystal structure reported here, resembles more closely the trimers in the T state enzyme than in the holoenzyme:bisubstrate-analog complex, which has been considered as the active, relaxed (R) state enzyme. Unlike the C trimer in either the T state or bisubstrate-analog-bound holoenzyme, the isolated C trimer lacks 3-fold symmetry, and the active sites are partially disordered. The flexibility of the C trimer, contrasted to the highly constrained T state ATCase, suggests that regulation of the holoenzyme involves modulating the potential for conformational changes essential for catalysis. Large differences in structure between the active C trimer and the holoenzyme:bisubstrate-analog complex call into question the view that this complex represents the activated R state of ATCase.
Bax, but not Bcl-x
L
, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations
Authors: Basañez G. , Nechushtan A. , Drozhinin O. , Chanturiya A. , Choe E. , Tutt S. , Wood K. A. , Hsu Y.-T. , Zimmerberg J. , Youle R. J.
Published: July 2002

Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-x
L
synthesized
in vitro
and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-x
L
, created arbitrary and continuously variable changes in membrane permeability and decreased the stability of the membrane, regardless of whether the source of the protein was synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage- and concentration-dependent manner. Bcl-x
L
did not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore—the apoptotic pore—large enough to allow mitochondrial proteins such as cytochrome
c
to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole.
Patterning cells and their environments using multiple laminar fluid flows in capillary networks
Authors: Takayama Shuichi , McDonald J. Cooper , Ostuni Emanuele , Liang Michael N. , Kenis Paul J. A. , Ismagilov Rustem F. , Whitesides George M.
Published: July 2002

This paper describes the use of laminar flow of liquids in capillary systems to pattern the cell culture substrate, to perform patterned cell deposition, and to pattern the cell culture media. We demonstrate the patterning of the cell culture substrate with different proteins, the patterning of different types of cells adjacent to each other, the patterned delivery of chemicals to adhered cells, and performing enzymatic reactions over select cells or over a portion of a cell. This method offers a way to simultaneously control the characteristics of the surface to which cells are attached, the type of cells that are in their vicinity, and the kind of media that cells or part of a cell are exposed to. The method is experimentally simple, highly adaptable, and requires no special equipment except for an elastomeric relief that can be readily prepared by rapid prototyping.
A functional bone morphogenetic protein system in the ovary
Authors: Shimasaki Shunichi , Zachow Rob J. , Li Danmei , Kim Holly , Iemura Shun-ichiro , Ueno Naoto , Sampath Kuber , Chang R. Jeffrey , Erickson Gregory F.
Published: July 2002

Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor β superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions.
In situ
hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought “luteinization inhibitor” in Graafian follicles during their growth and development.
Decoding photoperiodic time through
Per1
and ICER gene amplitude
Authors: Messager Sophie , Ross Alexander W. , Barrett Perry , Morgan Peter J.
Published: July 2002

The mammalian
Per1
gene is expressed in the suprachiasmatic nucleus of the hypothalamus, where it is thought to play a critical role in the generation of circadian rhythms.
Per1
mRNA also is expressed in other tissues. Its expression in the pars tuberalis (PT) of the pituitary is noteworthy because, like the suprachiasmatic nucleus, it is a known site of action of melatonin. The duration of the nocturnal melatonin signal encodes photoperiodic time, and many species use this to coordinate physiological adaptations with the yearly climatic cycle. This study reveals how the duration of photoperiodic time, conveyed through melatonin, is decoded as amplitude of
Per1
and ICER (inducible cAMP early repressor) gene expression in the PT. Syrian hamsters display a robust and transient peak of
Per1
and ICER gene expression 3 h after lights-on (Zeitgeber time 3) in the PT, under both long (16 h light/8 h dark) and short (8 h light/16 h dark) photoperiods. However, the amplitude of these peaks is greatly attenuated under a short photoperiod. The data show how amplitude of these genes may be important to the long-term measurement of photoperiodic time intervals.
BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation: A promising neuroprotective strategy
Authors: Chabrier Pierre-Etienne , Auguet Michel , Spinnewyn Brigitte , Auvin Serge , Cornet Sylvie , Demerlé-Pallardy Caroline , Guilmard-Favre Christine , Marin Jean-Grégoire , Pignol Bernadette , Gillard-Roubert Véronique , Roussillot-Charnet Christelle , Schulz Jocelyne , Viossat Isabelle , Bigg Dennis , Moncada Salvador
Published: July 2002

Nitric oxide (NO) and reactive oxygen species (ROS) act independently as well as cooperatively to induce neuronal death in acute neurological disorders. Inhibition of neuronal nitric oxide synthase (nNOS) and inhibition of lipid peroxidation induced by ROS have both been proposed as neuroprotective strategies in stroke and trauma. Recently, in our laboratory, the combination of the two strategies was found to be synergistic in reducing neuronal damage. Here, we report that BN 80933 [(
S
)-
N
-{4-[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2
H
-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl}-2-thiophenecarboximidamide], a compound that combines potent antioxidant and selective nNOS inhibitory properties
in vitro
, affords remarkable neuronal protection
in vivo
. Intravenous administration of BN 80933 significantly reduced brain damage induced by head trauma in mice, global ischemia in gerbils, and transient focal ischemia in rats. Treatment with BN 80933 (0.3–10 mg/kg) significantly reduced infarct volume (>60% protection) and enhanced behavioral recovery in rats subjected to transient (2-h) middle cerebral artery occlusion and 48-h or 7-day reperfusion. Furthermore, treatment with BN 80933 commencing up to 8 h after the onset of ischemia resulted in a significant improvement of neurological outcome. All these results indicate that BN 80933 represents a class of potentially useful therapeutic agents for the treatment of stroke or trauma and possibly neurodegenerative disorders that involve both NO and ROS.
Persistent membrane association of activated and depalmitoylated G protein α subunits
Authors: Huang Chunfa , Duncan Joseph A. , Gilman Alfred G. , Mumby Susanne M.
Published: July 2002

Heterotrimeric signal-transducing G proteins are organized at the inner surface of the plasma membrane, where they are positioned to interact with membrane-spanning receptors and appropriate effectors. G proteins are activated when they bind GTP and inactivated when they hydrolyze the nucleotide to GDP. However, the topological fate of activated G protein α subunits is disputed. One model declares that depalmitoylation of α, which accompanies activation by a receptor, promotes release of the protein into the cytoplasm. Our data suggest that activation of G protein α subunits causes them to concentrate in subdomains of the plasma membrane but not to be released from the membrane. Furthermore, α subunits remained bound to the membrane when they were activated with guanosine 5′-(3-
O
-thio)triphosphate and depalmitoylated with an acyl protein thioesterase. Limitation of α subunits to the plasma membrane obviously restricts their mobility and may contribute to the efficiency and specificity of signaling.
Generation of native bovine mAbs by phage display
Authors: O’Brien P. M. , Aitken R. , O’Neil B. W. , Campo M. S.
Published: July 2002

Modeling of disease pathogenesis and immunity often is carried out in large animals that are natural targets for pathogens of human or economic relevance. Although murine mAbs are a valuable tool in identifying certain host/pathogen interactions, progress in comparative immunology would be enhanced by the use of mAbs isolated from the host species. Such antibodies would reflect an authentic host immune response to infection or vaccination, and as they are host derived, would allow the application ofin vivoexperiments that previously have been unrealizable in large animals because of induction of an antispecies immune response. The advent of antibody phage display technology provides a way of producing host-derived mAbs in animals where the molecular genetics of Ig formation are known. Exploiting recent advances in the molecular immunology of cattle, we report here the design of an optimized phage display vector, pComBov, for the construction of combinatorial libraries of bovine Ig antigen-binding fragments (Fab) of native sequence. By using this system, we initially have generated and characterized a panel of bovine mAbs against a model antigen glutathioneS-transferase. The isolated mAbs showed features typical of bovine Igs and recognized glutathioneS-transferase with high specificity in ELISA and by Western blotting. The pComBov expression system can be readily adapted for the preparation of libraries from related ruminant species and advances the use of monoclonal reagents derived in this way for comparative studies in animals of economic importance.
CXCR4 utilization is sufficient to trigger CD4+ T cell depletion in HIV-1-infected human lymphoid tissue
Authors: Penn Michael L. , Grivel Jean-Charles , Schramm Birgit , Goldsmith Mark A. , Margolis Leonid
Published: July 2002

The human chemokine receptors CCR5 and CXCR4 have emerged as the predominant cofactors, along with CD4, for cellular entry of HIV-1in vivowhereas the contribution of other chemokine receptors to HIV disease has not been yet determined. CCR5-specific (R5) viruses predominate during primary HIV-1 infection whereas viruses with specificity for CXCR4 (R5/X4 or X4 viruses) often emerge in late stages of HIV disease. The evolution of X4 viruses is associated with a rapid decline in CD4+ T cells, although a causative relationship between viral tropism and CD4+ T cell depletion has not yet been proven. To rigorously test this relationship, we assessed CD4+ T cell depletion in suspensions of human peripheral blood mononuclear cells and in explants of human lymphoid tissue on exposure to paired viruses that are genetically identical (isogenic) except for select envelope determinants specifying reciprocal tropism for CXCR4 or CCR5. In both systems, X4 HIV-1 massively depleted CD4+ lymphocytes whereas matched R5 viruses depleted such cells only mildly despite comparable viral replication kinetics. These findings demonstrate that the coreceptor specificities of HIV-1 are a causal factor in CD4+ T cell depletionex vivoand strongly support the hypothesis that the evolution of viral envelope leading to usage of CXCR4in vivoaccelerates loss of CD4+ T cells, causing immunodeficiency.
Position effect of human telomeric repeats on replication timing
Authors: Ofir R. , Wong A. C. C. , McDermid H. E. , Skorecki K. L. , Selig S.
Published: July 2002

Telomeres are distinct structures, composed of short, repeated sequences, at the ends of all eukaryotic chromosomes. Telomeres have been shown in yeast to induce late replication in S phase and to silence transcription of neighboring genes. To examine the possibility of similar effects in human chromosomes, we studied cells from a subject with a microdeletion of 130 kb at the end of one copy of chromosome arm 22q, repaired by the addition of telomere repeats. Using fluorescencein situhybridization of S phase nuclei, a distinct difference was found in the replication timing of the breakpoint region between the intact and truncated copies of chromosome 22. This difference was evident as a shift from middle to late replication time of the breakpoint region adjacent to the repaired telomere. This finding suggests that the human telomere sequence influences activation of adjacent replication origin(s). The difference in replication timing between the two chromosomes was not associated with differences in sensitivity to digestion by DNase I or with methylation of regions immediately adjacent to the breakpoint. Furthermore, both alleles of arylsulfatase A, a gene located at a distance of approximately 54 kb from the breakpoint, were expressed. We conclude that as in yeast, the proximity of telomeric DNA may induce a positional effect that delays the replication of adjacent chromosomal regions in humans.
Identification ofMycobacterium tuberculosisRNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS)
Authors: Graham James E. , Clark-Curtiss Josephine E.
Published: July 2002

A widely applicable, positive cDNA selection method was developed to identify RNAs synthesized byMycobacterium tuberculosisin response to phagocytosis by cultured human primary macrophages. cDNAs forsigEandsigH(alternative sigma factors),aceA(isocitrate lyase),ponA(class I penicillin-binding protein),pks2(polyketide synthase),uvrA(UvrABC endonuclease), andctpV(putative cation transporter) were obtained from macrophage-grown bacteria. cDNAs for ORFs Rv3070, Rv3483c, Rv0903c (encoding a putative bacterial two-component transcriptional activator), and Rv0170 of themce1virulence operon also were obtained from phagocytized bacilli. cDNAs for these genomic regions were not obtained from approximately 1,000-fold more bacteria grown in laboratory broth. Methods described here, which have identifiedM. tuberculosisgenes expressed in response to host interaction, will allow the study of gene expression in a variety of microorganisms, including expression resulting from interaction with human tissues in natural disease states.
Impaired cerebellar synapse maturation in
waggler
, a mutant mouse with a disrupted neuronal calcium channel γ subunit
Authors: Chen Lu , Bao Shaowen , Qiao Xiaoxi , Thompson Richard F.
Published: July 2002

The
waggler
, a neurological mutant mouse with a disrupted putative neuronal Ca
2+
channel γ subunit, exhibits a cerebellar granule cell-specific brain-derived neurotrophic factor deficit, severe ataxia, and impaired eyeblink conditioning. Here, we show that multiple synapses of
waggler
cerebellar granule cells are arrested at an immature stage during development. Synaptic transmission is reduced at parallel fiber–Purkinje cell synapses. The Golgi cell–granule cell synaptic currents show immature kinetics associated with reduced γ-aminobutyric acid type A receptor α6 subunit expression in granule cells. In addition, the mossy fiber–granule cell synapses exhibit
N-
methyl-
d
-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs), but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSCs. Our results suggest that voltage-dependent Ca
2+
channels are involved in synapse maturation. This deficient synaptic transmission in the
waggler
cerebellum may account for their behavioral deficits.
hMutSα- and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage
Authors: Duckett Derek R. , Bronstein S. Maynard , Taya Yoichi , Modrich Paul
Published: July 2002

hMSH2⋅hMSH6 heterodimer (hMutSα) and hMLH1⋅hPMS2 complex (hMutLα) have been implicated in the cytotoxic response of mammalian cells to a number of DNA-damaging compounds, including methylating agents that produce
O
6
-methylguanine (O
6
MeG) adducts. This study demonstrates that O
6
MeG lesions, in which the damaged base is paired with either T or C, are subject to excision repair in a reaction that depends on a functional mismatch repair system. Furthermore, treatment of human cells with the S
N
1 DNA methylators
N
-methyl-
N
-nitrosourea or
N
-methyl-
N′
-nitro-
N
-nitrosoguanidine results in p53 phosphorylation on serine residues 15 and 392, and these phosphorylation events depend on the presence of functional hMutSα and hMutLα. Coupled with the previous demonstration that O
6
MeG⋅T and O
6
MeG⋅C pairs are recognized by hMutSα, these results implicate action of the mismatch repair system in the initial step of a damage-signaling cascade that can lead to cell-cycle checkpoint activation or cell death in response to DNA methylator damage.
SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor β signaling
Authors: Sun Yin , Liu Xuedong , Ng-Eaton Elinor , Lodish Harvey F. , Weinberg Robert A.
Published: July 2002

Transforming growth factor β (TGF-β) regulates a variety of physiologic processes, including growth inhibition, differentiation, and induction of apoptosis. Some TGF-β-initiated signals are conveyed through Smad3; TGF-β binding to its receptors induces phosphorylation of Smad3, which then migrates to the nucleus where it functions as a transcription factor. We describe here the association of Smad3 with the nuclear protooncogene protein SnoN. Overexpression of SnoN represses transcriptional activation by Smad3. Activation of TGF-β signaling leads to rapid degradation of SnoN and, to a lesser extent, of the related Ski protein, and this degradation is likely mediated by cellular proteasomes. These results demonstrate the existence of a cascade of the TGF-β signaling pathway, which, upon TGF-β stimulation, leads to the destruction of protooncoproteins that antagonize the activation of the TGF-β signaling.
cDNA microarrays detect activation of a myogenic transcription program by thePAX3-FKHRfusion oncogene
Authors: Khan Javed , Bittner Michael L. , Saal Lao H. , Teichmann Ulrike , Azorsa David O. , Gooden Gerald C. , Pavan William J. , Trent Jeffrey M. , Meltzer Paul S.
Published: July 2002

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion ofPAX3, a developmental transcription factor required for limb myogenesis, withFKHR, a member of the forkhead family of transcription factors. The resultantPAX3-FKHRgene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, bothPax3andPAX3-FKHRwere introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factorsMyoD, Myogenin,Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor geneIgf2and its binding proteinIgfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.
Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage
Authors: Klungland Arne , Rosewell Ian , Hollenbach Stephan , Larsen Elisabeth , Daly Graham , Epe Bernd , Seeberg Erling , Lindahl Tomas , Barnes Deborah E.
Published: July 2002

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G·C→T·A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the
OGG1
gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous
ogg1
−/−
null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of
ogg1
null mouse tissues cannot excise the damaged base, but there is significant slow removal
in vivo
from proliferating cells
.
These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.
CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells
Authors: Morales Janine , Homey Bernhard , Vicari Alain P. , Hudak Susan , Oldham Elizabeth , Hedrick Joseph , Orozco Rocio , Copeland Neal G. , Jenkins Nancy A. , McEvoy Leslie M. , Zlotnik Albert
Published: July 2002

In contrast to naive lymphocytes, memory/effector lymphocytes can access nonlymphoid effector sites and display restricted, often tissue-selective, migration behavior. The cutaneous lymphocyte-associated antigen (CLA) defines a subset of circulating memory T cells that selectively localize in cutaneous sites mediated in part by the interaction of CLA with its vascular ligand E-selectin. Here, we report the identification and characterization of a CC chemokine, cutaneous T cell-attracting chemokine (CTACK). Both human and mouse CTACK are detected only in skin by Southern and Northern blot analyses. Specifically, CTACK message is found in the mouse epidermis and in human keratinocytes, and anti-CTACK mAbs predominantly stain the epithelium. Finally, CTACK selectively attracts CLA+memory T cells. Taken together, these results suggest an important role for CTACK in recruitment of CLA+T cells to cutaneous sites. CTACK is predominantly expressed in the skin and selectively attracts a tissue-specific subpopulation of memory lymphocytes.
Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines
Authors: Shimizu K. , Fields R. C. , Giedlin M. , Mulé J. J.
Published: July 2002

We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses bothin vitroandin vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory/growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of nontoxic doses of recombinant IL-2 (20,000 and 40,000 IU/dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-γ production in these treated mice. Moreover, treatment with the combination of tumor lysate-pulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14- and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DCin vivoand provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.
Continuous axenic cultivation of
Pneumocystis carinii
Authors: Merali Salim , Frevert Ute , Williams Jonathan H. , Chin Kevin , Bryan Richard , Clarkson Allen B.
Published: July 2002

Continuous axenic culture of
Pneumocystis carinii
has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle’s salt supplemented with
S
-adenosyl-
l
-methionine, putrescine, ferric pyrophosphate,
N
-acetyl glucosamine, putrescine,
p
-aminobenzoic acid,
l
-cysteine and
l
-glutamine, and horse serum. Incubation is in room air at 31°C. The pH of the medium begins at 8.8 and rises to ≈9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of
P. carinii
, and
P. carinii
-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture.
Cytoplasmic RNA modulators of an inside-out signal-transduction cascade
Authors: Blind Michael , Kolanus Waldemar , Famulok Michael
Published: July 2002

A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the β2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathwaysin vivocould potentially be applied wherever targeted modulation of a signal-transduction cascade is desired.
Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening
Authors: Zhu Jiyue , Wang He , Bishop J. Michael , Blackburn Elizabeth H.
Published: July 2002

Human fibroblasts whose lifespan in culture has been extended by expression of a viral oncogene eventually undergo a growth crisis marked by failure to proliferate. It has been proposed that telomere shortening in these cells is the property that limits their proliferation. Here we report that ectopic expression of the wild-type reverse transcriptase protein (hTERT) of human telomerase averts crisis, at the same time reducing the frequency of dicentric and abnormal chromosomes. Surprisingly, as the resulting immortalized cells containing active telomerase continue to proliferate, their telomeres continue to shorten to mean lengths below those in control cells that enter crisis. These results provide evidence for a protective function of human telomerase that allows cell proliferation without requiring net lengthening of telomeres.
The intracellular domain of interferon-α receptor 2c (IFN-αR2c) chain is responsible for Stat activation
Authors: Kotenko Serguei V. , Izotova Lara S. , Mirochnitchenko Olga V. , Lee Carolyn , Pestka Sidney
Published: July 2002

Type I IFNs activate the Jak–Stat signal transduction pathway. The IFN-α receptor 1 (IFN-αR1) subunit and two splice variants of the IFN-αR2 subunit, IFN-αR2c and IFN-αR2b, are involved in ligand binding. All these receptors have been implicated in cytokine signaling and, specifically, in Stat recruitment. To evaluate the specific contribution of each receptor subunit to Stat recruitment we employed chimeric receptors with the extracellular domain of either IFN-γR2 or IFN-γR1 fused to the intracellular domains of IFN-αR1, IFN-αR2b, and IFN-αR2c. These chimeric receptors were expressed in hamster cells. Because human IFN-γ exhibits no activity on hamster cells, the use of the human IFN-γ receptor extracellular domains allowed us to avoid the variable cross-species activity of the type I IFNs and eliminate the possibility of contributions of endogenous type I IFN receptors into the Stat recruitment process. We demonstrate that Stat recruitment is solely a function of the IFN-αR2c intracellular domain. When chimeric receptors with the human IFN-γR1 extracellular domain and various human IFN-α receptor intracellular domains were expressed in hamster cells carrying the human IFN-γR2 subunit, only the IFN-αR2c subunit was capable of supporting IFN-γ signaling as measured by MHC class I induction, antiviral protection, and Stat activation. Neither the IFN-αR2b nor the IFN-αR1 intracellular domain was able to recruit Stats or support IFN-γ-induced biological activities. Thus, the IFN-αR2c intracellular domain is necessary and sufficient to activate Stat1, Stat2, and Stat3 proteins.
Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein
Authors: Pearce Linda L. , Gandley Robin E. , Han Weiping , Wasserloos Karla , Stitt Molly , Kanai Anthony J. , McLaughlin Margaret K. , Pitt Bruce R. , Levitan Edwin S.
Published: July 2002

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from
in vitro
studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs).
In vitro
studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca
2+
act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT
−/−
) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.
Cloning and characterization of the mammalian brain-specific, Mg
2+
-dependent neutral sphingomyelinase
Authors: Hofmann Kay , Tomiuk Stefan , Wolff Gabriela , Stoffel Wilhelm
Published: July 2002

The enzymatic breakdown of sphingomyelin by sphingomyelinases is considered the major source of the second messenger ceramide. Studies on the contribution of the various described acidic and neutral sphingomyelinases to the signaling pool of ceramide have been hampered by the lack of molecular data on the neutral sphingomyelinases (nSMases). We recently identified a mammalian nSMase, an integral membrane protein with remote similarity to bacterial sphingomyelinases. However, its ubiquitous expression pattern is in contrast to previous findings that sphingomyelinase activity is found mainly in brain tissues. By using an improved database search method, combined with phylogenetic analysis, we identified a second mammalian nSMase (nSMase2) with predominant expression in the brain. The sphingomyelinase activity of nSMase2 has a neutral pH optimum, depends on Mg
2+
ions, and is activated by unsaturated fatty acids and phosphatidylserine. Immunofluorescence reveals a neuron-specific punctate perinuclear staining, which colocalizes with a Golgi marker in a number of cell lines. The likely identity of nSMase2 with cca1, a rat protein involved in contact inhibition of 3Y1 fibroblasts, suggests a role for this enzyme in cell cycle arrest. Both mammalian nSMases are members of a superfamily of Mg
2+
-dependent phosphohydrolases, which also contains nucleases, inositol phosphatases, and bacterial toxins.
Reproductive systems and evolution in vascular plants
Authors: Holsinger Kent E.
Published: July 2002

Differences in the frequency with which offspring are produced asexually, through self-fertilization and through sexual outcrossing, are a predominant influence on the genetic structure of plant populations. Selfers and asexuals have fewer genotypes within populations than outcrossers with similar allele frequencies, and more genetic diversity in selfers and asexuals is a result of differences among populations than in sexual outcrossers. As a result of reduced levels of diversity, selfers and asexuals may be less able to respond adaptively to changing environments, and because genotypes are not mixed across family lineages, their populations may accumulate deleterious mutations more rapidly. Such differences suggest that selfing and asexual lineages may be evolutionarily short-lived and could explain why they often seem to be of recent origin. Nonetheless, the origin and maintenance of different reproductive modes must be linked to individual-level properties of survival and reproduction. Sexual outcrossers suffer from a cost of outcrossing that arises because they do not contribute to selfed or asexual progeny, whereas selfers and asexuals may contribute to outcrossed progeny. Selfing and asexual reproduction also may allow reproduction when circumstances reduce opportunities for a union of gametes produced by different individuals, a phenomenon known as reproductive assurance. Both the cost of outcrossing and reproductive assurance lead to an over-representation of selfers and asexuals in newly formed progeny, and unless sexual outcrossers are more likely to survive and reproduce, they eventually will be displaced from populations in which a selfing or asexual variant arises.
Mushroom body defect
, a gene involved in the control of neuroblast proliferation in
Drosophila
, encodes a coiled–coil protein
Authors: Guan Zhonghui , Prado Antonio , Melzig Jörg , Heisenberg Martin , Nash Howard A. , Raabe Thomas
Published: July 2002

Neurogenesis relies on the establishment of the proper number and precisely controlled proliferation of neuroblasts, the neuronal precursor cells. A role for the
mushroom body defect
(
mud
) gene in both of these aspects of neuroblast behavior, as well as possible roles in other aspects of fruit fly biology, is implied by phenotypes associated with
mud
mutations. We have localized
mud
by determining the sequence change in one point mutant, identifying a predicted ORF affected by the mutation, and showing that an appropriate segment of the genome rescues
mud
mutant phenotypes. An analysis of
mud
cDNAs and a survey of
mud
transcripts by Northern blotting indicate that the gene is subject to differential splicing and is expressed primarily during embryogenesis but also, at lower levels, during subsequent developmental stages in a sexually dimorphic manner. The gene is predicted to encode a polypeptide without obvious homologs but with two prominent structural features, a long coiled coil that constitutes the central core of the protein and a carboxyl-terminal transmembrane domain.
Interpreting the universal phylogenetic tree
Authors: Woese Carl R.
Published: July 2002

The universal phylogenetic tree not only spans all extant life, but its root and earliest branchings represent stages in the evolutionary process before modern cell types had come into being. The evolution of the cell is an interplay between vertically derived and horizontally acquired variation. Primitive cellular entities were necessarily simpler and more modular in design than are modern cells. Consequently, horizontal gene transfer early on was pervasive, dominating the evolutionary dynamic. The root of the universal phylogenetic tree represents the first stage in cellular evolution when the evolving cell became sufficiently integrated and stable to the erosive effects of horizontal gene transfer that true organismal lineages could exist.
Specific chromosome 1 breaks induced by human cytomegalovirus
Authors: Fortunato Elizabeth A. , Dell'Aquila Marie L. , Spector Deborah H.
Published: July 2002

Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem for immunocompromised individuals. Here we show that infection of cells with HCMV during the S-phase of the cell cycle results in two specific chromosome 1 breaks at positions 1q42 and 1q21. We demonstrate that purified virions, and not infected cell supernatant alone, are responsible for the damage. In addition, we show that the specific breaks occur when different sources of fibroblasts and strains of HCMV are used. Incubation of the virus with neutralizing antibody prevents the induction of breaks. However, UV-inactivated virus is as efficient as untreated virus in inducing specific damage to chromosome 1. Thus, there is a requirement for viral adsorption/penetration, but not new viral gene expression. This HCMV-mediated induction of site-specific damage in actively dividing cells may provide clues for the development of neurological defects in the congenitally infected infant.