bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell type-specific loss of
atp6
RNA editing in cytoplasmic male sterile
Sorghum bicolor
Authors: Howad Werner , Kempken Frank
Published: July 2002

RNA editing and cytoplasmic male sterility are two important phenomena in higher plant mitochondria. To determine whether correlations might exist between the two, RNA editing in different tissues of
Sorghum bicolor
was compared employing reverse transcription–PCR and subsequent sequence analysis. In etiolated shoots, RNA editing of transcripts of plant mitochondrial
atp6
,
atp9
,
nad3
,
nad4
, and
rps12
genes was identical among fertile or cytoplasmic male sterile plants. We then established a protocol for mitochondrial RNA isolation from plant anthers and pollen to include in these studies. Whereas RNA editing of
atp9
,
nad3
,
nad4
, and
rps12
transcripts in anthers was similar to etiolated shoots, mitochondrial
atp6
RNA editing was strongly reduced in anthers of the A3Tx398 male sterile line of
S. bicolor
.
atp6
transcripts of wheat and selected plastid transcripts in
S. bicolor
showed normal RNA editing, indicating that loss of
atp6
RNA editing is specific for cytoplasmic male sterility
S. bicolor
mitochondria. Restoration of fertility in F1 and F2 lines correlated with an increase in RNA editing of
atp6
transcripts. Our data suggest that loss of
atp6
RNA editing contributes to or causes cytoplasmic male sterility in
S. bicolor
. Further analysis of the mechanism of cell type-specific loss of
atp6
RNA editing activity may advance our understanding of the mechanism of RNA editing.
The Tabby phenotype is caused by mutation in a mouse homologue of theEDAgene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains
Authors: Srivastava Anand K. , Pispa Johanna , Hartung Andrew J. , Du Yangzhu , Ezer Sini , Jenks Ted , Shimada Tokihiko , Pekkanen Maija , Mikkola Marja L. , Ko Minoru S. H. , Thesleff Irma , Kere Juha , Schlessinger David
Published: July 2002

Mouse Tabby (Ta) and X chromosome-linked humanEDAshare the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned theTagene and find it to be homologous to theEDAgene. The gene is altered in twoTaalleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues fromTamice.TaandEDAgenes both encode alternatively spliced forms; novel exons now extend the 3′ end of theEDAgene. All transcripts recovered have the same 5′ exon. The longestTacDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development.
A resource range invariance rule for optimal offspring size predicts patterns of variability in parental phenotypes
Authors: Downhower Jerry F. , Charnov Eric L.
Published: July 2002

Previous analysis of the rules regarding how much more a female should invest in a litter of size C rather than producing a litter with one more offspring revealed an invariance relationship between litter size and the range of resources per offspring in any litter size. The rule is that the range of resources per offspring should be inversely proportional to litter size. Here we present a modification of this rule that relates litter size to the total resources devoted to reproduction at that litter size. The result is that the range of resources devoted to reproduction should be the same for all litter sizes. When parental phenotypes covary linearly with resources devoted to reproduction, then those traits should also show equal ranges within each litter size category (except for litters of one). We tested this prediction by examining the range in body size (=total length) of female mosquito fish (Gambusia hubbsi) at different litter sizes. Because resources devoted to reproduction may take many forms (e.g., nest defense), this prediction may have broad applicability.
Synaptic facilitation by reflected action potentials: Enhancement of transmission when nerve impulses reverse direction at axon branch points
Authors: Baccus Stephen A.
Published: July 2002

A rapid, reversible enhancement of synaptic transmission from a sensory neuron is reported and explained by impulses that reverse direction, or reflect, at axon branch points. In leech mechanosensory neurons, where one can detect reflection because it is possible simultaneously to study electrical activity in separate branches, action potentials reflected from branch points within the central nervous system under physiological conditions. Synapses adjacent to these branch points were activated twice in rapid succession, first by an impulse arriving from the periphery and then by its reflection. This fast double-firing facilitated synaptic transmission, increasing it to more than twice its normal level. Reflection occurred within a range of resting membrane potentials, and electrical activity produced by mechanical stimulation changed membrane potential so as to produce and cease reflection. A compartmental model was used to investigate how branch-point morphology and electrical activity contribute to produce reflection. The model shows that mechanisms that hyperpolarize the membrane so as to impair action potential propagation can increase the range of structures that can produce reflection. This suggests that reflection is more likely to occur in other structures where impulses fail, such as in axons and dendrites in the mammalian brain. In leech sensory neurons, reflection increased transmission from central synapses only in those axon branches that innervate the edges of the receptive field in the skin, thereby sharpening spatial contrast. Reflection thus allows a neuron to amplify synaptic transmission from a selected group of its branches in a way that can be regulated by electrical activity.
Mortality invariants and their genetic implications
Authors: Azbel’ Mark Yacov
Published: July 2002

Old noninbred fly mortality decreases according to the inverse linear law and reduces to a single suborder-specific age. Relative child mortality (the mortality at a given age related to the mortality at 10 years) from 1 mo to 11 years is the same with 8% mean accuracy for all humans, independent of race, country, sex, and birth year (from 1780 to 1995), in contrast to birth mortality, which in developed countries changed fiftyfold during the last century. The concept of invariants, which is very powerful in physics, is applied to mortality of species as remote as humans and flies. It provides quantitative estimates for the selection of hereditary Methuselahs, who live, e.g., over six-mean lifespans and who may be relatively young biologically. It also demonstrates that old fly and relative child mortality are determined genetically and that the former is related to genetic heterogeneity.
From differentiation to proliferation: The secretory amyloid precursor protein as a local mediator of growth in thyroid epithelial cells
Authors: Pietrzik Claus Ulrich , Hoffmann Jens , Stöber Kai , Chen Chun-Yan , Bauer Christoph , Otero Deborah A. C. , Roch Jean-Marc , Herzog Volker
Published: July 2002

In various species, thyrotropin (TSH) is known to stimulate both differentiation and proliferation of thyroid follicle cells. This cell type has also been shown to express members of the Alzheimer amyloid precursor (APP) protein family and to release the secretory N-terminal domain of APP (sAPP) in a TSH-dependent fashion. In this study on binding to the cell surfaces, exogenously added recombinant sAPP stimulated phosphorylation mediated by mitogen-activated protein kinase and effectively evoked proliferation in the rat thyroid epithelial cell line FRTL-5. To see whether this proliverative effect of sAPP is of physiological relevance, we used antisense techniques to selectively inhibit the expression of APP and the proteolytic release of sAPP by cells grown in the presence of TSH. The antisense-induced inhibition was detected by immunoblot, immunoprecipitation, and immunocytochemical analyses. After the reduced APP expression and sAPP secretion, we observed a strong suppression of the TSH-induced cell proliferation down to 35%. Recombinant sAPP but not TSH was able to overcome this antisense effect and to completely restore cell proliferation, indicating that sAPP acts downstream of TSH, in that it is released from thyroid epithelial cells during TSH-induced differentiation. We propose that sAPP operates as an autocrine growth factor mediating the proliferative effect of TSH on neighboring thyroid epithelial cells.
Movement of rice yellow mottle virus between xylem cells through pit membranes
Authors: Opalka Natacha , Brugidou Christophe , Bonneau Caroline , Nicole Michel , Beachy Roger N. , Yeager Mark , Fauquet Claude
Published: July 2002

The translocation of rice yellow mottle virus (RYMV) within tissues of inoculated and systemically infected
Oryza sativa
L. leaves was characterized by Western immunoblotting, Northern blotting, and electron microscopy of thin sections. In inoculated leaves, RYMV RNA and coat protein first were detected at 3 and 5 days postinoculation, respectively. By 6 days postinoculation, RYMV had spread systemically to leaves, and virus particles were observed in most cell types, including epidermal, mesophyll, bundle sheath, and vascular parenchyma cells. Most of the virions accumulated in large crystalline patches in xylem parenchyma cells and sieve elements. Colocalization of a cell wall marker for cellulosic β-(1–4)-
d
-glucans and anti-RYMV antibodies over vessel pit membranes suggests a pathway for virus migration between vessels. We propose that the partial digestion of pit membranes resulting from programmed cell death may permit virus migration through them, concomitant with autolysis. In addition, displacement of the Ca
2+
from pit membranes to virus particles may contribute to the disruption of the pit membranes and facilitate systemic virus transport.
In search of planets and life around other stars
Authors: Lunine Jonathan I.
Published: July 2002

The discovery of over a dozen low-mass companions to nearby stars has intensified scientific and public interest in a longer term search for habitable planets like our own. However, the nature of the detected companions, and in particular whether they resemble Jupiter in properties and origin, remains undetermined.
Bypassing the periplasm: Reconstitution of the AcrAB multidrug efflux pump of
Escherichia coli
Authors: Zgurskaya Helen I. , Nikaido Hiroshi
Published: July 2002

AcrAB is a constitutively expressed, major multidrug efflux system of
Escherichia coli
. We have purified the cytoplasmic membrane component, AcrB, to near homogeneity, and reconstituted the protein into proteoliposomes. In the presence of ΔpH (outside acid), the protein catalyzed the extrusion of fluorescent phospholipids, which were then trapped by protein-free acceptor vesicles. Known substrates of AcrAB, such as bile acids, erythromycin, and cloxacillin, inhibited this activity. Addition of various drugs to AcrB-containing proteoliposomes, in the presence of ΔpH (inside acid) resulted in proton efflux, suggesting that AcrB is a proton antiporter. Interestingly, fluorescent lipid extrusion was accelerated strongly by the periplasmic protein AcrA in the presence of Mg
2+
, and at pH 5.0 AcrA alone produced a slow mixing of lipids of different vesicles, without causing the mixing of intravesicular material. These results suggest that AcrA brings two membranes together, and under certain conditions may even cause the fusion of at least the outer leaflets of the membranes, contributing to the ability of the AcrAB–TolC system to pump drugs out directly into the medium.
Estrogen-inducible, sex-specific expression of brain-derived neurotrophic factor mRNA in a forebrain song control nucleus of the juvenile zebra finch
Authors: Dittrich Falk , Feng Yu , Metzdorf Reinhold , Gahr Manfred
Published: July 2002

The expression of brain-derived neurotrophic factor (BDNF) mRNA is increased significantly within the high vocal center (HVc) of male but not female zebra finches from posthatching day 30–35 on. The population of HVc cells expressing BDNF mRNA included 35% of the neurons projecting to the nucleus robustus of the archistriatum (RA). In the RA and in RA-projecting neurons of the lateral portion of the magnocellular nucleus of the anterior neostriatum, BDNF mRNA was expressed at very low levels in both sexes. The BDNF-receptor trkB mRNA was expressed in the RA, in RA-projecting neurons of lateral portion of the magnocellular nucleus of the anterior neostriatum, and in the HVc, except in most of its RA-projecting neurons. Premature stimulation and an inhibitory effect on the normal increase of the BDNF mRNA expression in juvenile males occurred after treatments with 17β-estradiol and the aromatase inhibitor fadrozole, respectively. The up-regulation of the BDNF expression in the HVc could be a mechanism by which estrogen triggers the differentiation of cells within and connected to the HVc of male zebra finches.
Antiglucocorticoid activity of hepatocyte nuclear factor-6
Authors: Pierreux Christophe E. , Stafford John , Demonte Dominique , Scott Donald K. , Vandenhaute Jean , O’Brien Richard M. , Granner Daryl K. , Rousseau Guy G. , Lemaigre Frederic P.
Published: July 2002

Glucocorticoids exert their effects on gene transcription through ubiquitous receptors that bind to regulatory sequences present in many genes. These glucocorticoid receptors are present in all cell types, yet glucocorticoid action is controlled in a tissue-specific way. One mechanism for this control relies on tissue-specific transcriptional activators that bind in the vicinity of the glucocorticoid receptor and are required for receptor action. We now describe a gene-specific and tissue-specific inhibitory mechanism through which glucocorticoid action is repressed by a tissue-restricted transcription factor, hepatocyte nuclear factor-6 (HNF-6). HNF-6 inhibits the glucocorticoid-induced stimulation of two genes coding for enzymes of liver glucose metabolism, namely 6-phosphofructo-2-kinase and phospho
enol
pyruvate carboxykinase. Binding of HNF-6 to DNA is required for inhibition of glucocorticoid receptor activity.
In vitro
and
in vivo
experiments suggest that this inhibition is mediated by a direct HNF-6/glucocorticoid receptor interaction involving the amino-terminal domain of HNF-6 and the DNA-binding domain of the receptor. Thus, in addition to its known property of stimulating transcription of liver-expressed genes, HNF-6 can antagonize glucocorticoid-stimulated gene transcription.
Predominant VHgenes expressed in innate antibodies are associated with distinctive antigen-binding sites
Authors: Seidl Katherine J. , Wilshire Jennifer A. , MacKenzie John D. , Kantor Aaron B. , Herzenberg Leonard A. , Herzenberg Leonore A.
Published: July 2002

Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VHgene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJHrearrangements (CDR3, JH) associated with each VHgene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VHframework, is sufficient to define the specificity of an antibody.
The biological clock of very premature primate infants is responsive to light
Authors: Hao Haiping , Rivkees Scott A.
Published: July 2002

Each year more than 250,000 infants in the United States are exposed to artificial lighting in hospital nurseries with little consideration given to environmental lighting cycles. Essential in determining whether environmental lighting cycles need to be considered in hospital nurseries is identifying when the infant’s endogenous circadian clock becomes responsive to light. Using a non-human primate model of the developing human, we examined when the circadian clock, located in the hypothalamic suprachiasmatic nuclei (SCN), becomes responsive to light. Preterm infant baboons of different ages were exposed to light (5,000 lux) at night, and then changes in SCN metabolic activity and gene expression were assessed. After exposure to bright light at night, robust increases in SCN metabolic activity and gene expression were seen at ages that were equivalent to human infants at 24 weeks after conception. These data provide direct evidence that the biological clock of very premature primate infants is responsive to light.
Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1
Authors: Pereira Lygia , Lee Sui Ying , Gayraud Barbara , Andrikopoulos Kostantinos , Shapiro Steven D. , Bunton Tracie , Biery Nancy Jensen , Dietz Harry C. , Sakai Lynn Y. , Ramirez Francesco
Published: July 2002

Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory–fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.
AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation
Authors: Miyamoto Toshihiro , Weissman Irving L. , Akashi Koichi
Published: July 2002

Leukemia-specific
AML1/ETO
transcripts are detectable in most patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. To understand the inconsistency between the clinical cure and the presence of “residual disease” at a molecular level, we separated and identified the cells expressing AML1/ETO by phenotype and function. Here we demonstrate that
AML1/ETO
transcripts are present in a fraction of stem cells, monocytes, and B cells in remission marrow, and in a fraction of B cells in leukemic marrow, but not in T cells.
AML1/ETO
transcripts also were demonstrated in a fraction of colony-forming cells of erythroid, granulocyte-macrophage, and/or megakaryocyte lineages in both leukemic and remission marrow. These data strongly suggest that the acquisition of the t(8;21) occurs at the level of stem cells capable of differentiating into B cells as well as all myeloid lineages, and that a fraction of the AML1/ETO-expressing stem cells undergo additional oncogenic event(s) that ultimately leads to transformation into AML.
The coevolution of cell senescence and diploid sexual reproduction in unicellular organisms
Authors: Cui Yan , Chen Run Sheng , Wong Wing Hung
Published: July 2002

In this paper, we investigate the coevolution of diploid sexual reproduction and cell senescence (i.e., cell aging). We use probability analysis, computer simulation, and exact numerical computation to analyze the impacts of deleterious recessive mutations on sexual and asexual reproduction. The main conclusion is that, without cell senescence, the evolutionary advantages of sexual reproduction cannot be realized in unicellular organisms that reproduce both sexually and asexually. Also, cell senescence is found to be useful in the maintenance of sexual reproduction. This result suggests that diploid sexual reproduction was unlikely to establish itself as a widespread reproduction mechanism without the complementary process of cell senescence.
High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1
Authors: McNeish John , Aiello Robert J. , Guyot Deborah , Turi Tom , Gabel Christopher , Aldinger Charles , Hoppe Kenneth L. , Roach Marsha L. , Royer Lori J. , de Wet Jeffery , Broccardo Cyril , Chimini Giovanna , Francone Omar L.
Published: July 2002

Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental
in vivo
model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine
Abc1
gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from
Abc1
knockout (−/−) mice revealed an ≈70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/− and −/− compared with their littermate controls. The inactivation of the
Abc1
gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of
Abc1
−/− mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that
Abc1
−/− mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.
Similarities and Variations in Facial Expressions of Emotions: Cross‐cultural Evidence
Authors: Mandal Manas K. , Bryden M.P. , Bulman‐Fleming M.B.
Published: July 2002

North American (Canadian) and Indian observers were shown photographs of six facial emotions; happiness, sadness, fear, anger, surprise, and disgust, expressed by American Caucasian and Indian subjects. Observers were asked to judge each photograph, on a 7‐point scale, for the degree of (a) distinctiveness (free from blending with other emotion categories), (b) pleasantness‐unpleasantness, and (c) arousal‐nonarousal of expressed facial emotion. The results showed significant interaction of Observer × Expressor × Emotion for the distinctiveness judgement. It was found that fearful and angry expressions in Indian faces, in comparison to Caucasian faces, were judged as less distinctly identifiable by observers of both cultural origins. Indian observers rated these two emotion expressions as being more distinctive than did North Americans irrespective of the culture of the expressor. In addition, Indian observers judged fearful and angry expressions as more unpleasant than did North Americans. Caucasians, in comparison to Indians, were judged to have more arousal in most of the emotion expressions.
The Gypsies of Romania Since 1990*
Authors: Crowe David M.
Published: July 2002

The Roma or Gypsies entered Romania's historic provinces, Wallachia and Moldavia, in the twelfth century. Over the next 200 years, the Roma, who had come to the Balkans from northern India, were enslaved. By the fifteenth century, the practice of Gypsy slavery was widespread throughout the two provinces. In part, their enslavement came about as a means of securing Gypsy skills as craftsmen, metalsmiths, musicians, and equine specialists. Over time, a complex body of laws was passed in Wallachia and Moldavia to strengthen the control of Romanian noblemen over their Gypsy slaves (robi). However, by the eighteenth century, some mild efforts were undertaken to better the plight of Romanian Gypsy slaves.
Neural Activity Related to Anger in Cocaine‐Dependent Men: A Possible Link to Violence and Relapse
Authors: Drexler Karen , Schweitzer Julie B. , Quinn Colin K. , Gross Robin , Ely Timothy D. , Muhammad Faheemah , Kilts Clinton D.
Published: July 2002

This study examined the neural correlates of cue‐induced anger in cocaine‐dependent men in an initial investigation of possible neurobiological explanations for the putative association between cocaine addiction and violence. We used positron emission tomography (PET) to localize alterations in regional cerebral blood flow (rCBF) during mental imagery of a personal anger‐associated scene and of an emotionally neutral scene in ten cocaine‐dependent men. Compared to the emotionally neutral imagery control condition, anger was associated with marked decreases in rCBF in multiple areas of the frontal cortex (particularly the right inferior frontal gyrus), left posterior insula, left fusiform gyrus, and midbrain. Conversely, this same inferior frontal area was activated by anger imagery in nicotine‐dependent men. Anger was also associated with increases in rCBF in the right fusiform gyrus, right and left middle occipital gyri, left postcentral gyrus, left medial frontal gyrus, left cuneus, and in the left anterior cingulate gyrus. The study showed that cue‐induced anger in cocaine‐dependent men was associated with decreased activity in frontal cortical areas involved in response monitoring and inhibition. The lack of this association in nicotine‐dependent men suggests a possible deficit in anger regulation associated with cocaine dependence and a possible link between cocaine dependence, violence, and relapse.
Gender-Based Differences in Rats after Chronic Dietary Exposure to Genistein
Authors: Slikker W. , Scallet A. C. , Doerge D. R. , Ferguson S. A.
Published: July 2002

Gender-based differences can be observed from pharmacokinetic, behavioral, or anatomical assessments. No single assessment tool will provide a complete answer, but the use of a variety of indices, each with known gender-related outcome differences, can reveal agent-induced gender-based alterations. In a series of initial range-finding studies in rats conducted at the National Center for Toxicological Research (NCTR), the effects of dietary exposure to the weak estrogen, genistein, have been assessed using a number of techniques with validated gender-related outcome measures. The findings indicated that (1) the internal dose of genistein was higher in females than males after equivalent dietary exposure and this was consistent with the faster rate of genistein elimination in males; (2) in behavioral assessments, males and females in the high-dose dietary genistein group consumed more of a sodium-flavored solution; however, no genistein-related changes were observed in open field or running wheel activity, play behavior, or intake of a saccharin-flavored solution; and (3) dose-related alterations of the volume of the sexually dimorphic nucleus of the medial preoptic area were observed in genistein-exposed male rats but not females. These observations describe the utility of a variety of gender-based assessment tools and indicate that dose-related effects of developmental and chronic dietary exposure to genistein can be observed in the rodent. Additional studies, perhaps in nonhuman primates, are necessary to further predict the effect(s) of genistein on human gender-based development.
Oral and Dermal Administration of Clarified Slurry Oil to Male C3H Mice
Authors: Feuston M. H. , Hamilton C. E. , Mackerer C. R.
Published: July 2002

Clarified slurry oil (CSO) is the heavy residual material produced in the fluidized catalytic cracking unit of a petroleum refinery. CSO was adm in istered via gavage or dermal application to male C3H mice (10/ group) at a dose of 1000 mg/ kg/ d, 5 d/ wk, for up to 10 weeks. Interim sacrifices were performed after 2, 4, 8, and 10 weeks of treatment for orally exposed and control mice; dermally exposed mice were sacrificed after 10 weeks. Fifty percent of the “dermal” mice died prior to scheduled sacrifice. Deaths observed in the “oral” group were attributed to apparent misintubations, not to CSO exposure. Mice exposed orally for 2, 4, or 8 weeks (but not 10 weeks) exhibited significantly increased absolute (A) and relative (R) liver weights, while all “oral” mice (including those sacrificed after 10 weeks) had significantly decreased A and R thymus weights when compared to controls. “Dermal” mice had significantly increased R liver weights and significantly decreased A and R thymus weights. After 2 weeks most “oral” mice showed hepatocyte hypertrophy. A few mice showed liver necrosis. Liver findings increased in incidence and severity until 8 weeks of exposure. After 10 weeks, the liver of most “oral” mice showed the same or less discern ible morphologic pathology than was observed after 2 weeks. At 10 weeks, “dermal” mice showed the previously described liver effects, except that liver necrosis was severe, and evidence of fibrosis was observed. In summary, although CSO was hepatotoxic, “oral” mice showed signs of liver recovery after 10 weeks of exposure despite continued treatment. Thus, CSO appears to be more toxic to mice when administered dermally than orally. Humans are more likely to come into contact with these lipids by dermal rather than by oral exposure.
Chicken Acidic Leucine-rich EGF-like Domain Containing Brain Protein (CALEB), a Neural Member of the EGF Family of Differentiation Factors, Is Implicated in Neurite Formation
Authors: Schumacher Stefan , Volkmer Hansjürgen , Buck Fritz , Otto Albrecht , Tárnok Attila , Roth Siegfried , Rathjen Fritz G.
Published: July 2002

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline–rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.
Ran-unassisted Nuclear Migration of a 97-kD Component of Nuclear Pore–targeting Complex
Authors: Kose Shingo , Imamoto Naoko , Tachibana Taro , Shimamoto Takuya , Yoneda Yoshihiro
Published: July 2002

A 97-kD component of nuclear pore-targeting complex (the β-subunit of nuclear pore–targeting complex [PTAC]/importin/karyopherin) mediates the import of nuclear localization signal (NLS)-containing proteins by anchoring the NLS receptor protein (the α-subunit of PTAC/importin/karyopherin) to the nuclear pore complex (NPC). The import requires a small GTPase Ran, which interacts directly with the β-subunit. The present study describes an examination of the behavior of the β-subunit in living cells and in digitonin-permeabilized cells. In living cells, cytoplasmically injected β-subunit rapidly migrates into the nucleus. The use of deletion mutants reveals that nuclear migration of the β-subunit requires neither Ran- nor α-subunit–binding but only the NPC-binding domain of this molecule, which is also involved in NLS-mediated import. Furthermore, unlike NLS-mediated import, a dominant-negative Ran, defective in GTP-hydrolysis, did not inhibit nuclear migration of the β-subunit. In the digitonin-permeabilized cell-free import assay, the β-subunit transits rapidly through the NPC into the nucleus in a saturating manner in the absence of exogenous addition of soluble factors. These results show that the β-subunit undergoes translocation at the NPC in a Ran-unassisted manner when it does not carry α-subunit/NLS substrate. Therefore, a requirement for Ran arises only when the β-subunit undergoes a translocation reaction together with the α-subunit/NLS substrate. The results provide an insight to the yet unsolved question regarding the mechanism by which proteins are directionally transported through the NPC, and the role of Ran in this process.
Extracellular ATP Activates Transcription Factor NF-κB through the P2Z Purinoreceptor by Selectively Targeting NF-κB p65 (RelA)
Authors: Ferrari Davide , Wesselborg Sebastian , Bauer Manuel K.A. , Schulze-Osthoff Klaus
Published: July 2002

Cells of the macrophage lineage express a peculiar surface receptor for extracellular ATP, designated P2Z/P2X7 purinergic receptor, that induces pore formation and collapse of the plasma membrane potential. Although the function of the P2Z receptor is largely unknown, accumulating evidence implicates its role in cell signaling and immune reactions. Here, we investigated the effect of P2Z receptor ligation on the activation of NF-κB, a transcription factor controlling cytokine expression and apoptosis. Exposure of microglial cells to ATP but not other nucleotides resulted in potent NF-κB activation. This effect was specifically mediated by the P2Z receptor, because selective receptor antagonists prevented NF-κB activation. NF-κB activation required reactive oxygen intermediates and proteases of the caspase family, because it was abolished by antioxidants and specific protease inhibitors. The subunit composition of the ATP-induced NF- κB–DNA complex was rather unusual. Whereas exposure to LPS-induced prototypical NF-κB p50 homo- and p65 (RelA)/p50 heterodimers, ATP stimulation resulted in the sole appearance of a p65 homodimer. This is the first demonstration that a certain stimulus activates a particular NF-κB subunit. Because different NF-κB complexes exhibit distinct transcriptional and DNA-binding activities, ATP may control the expression of a subset of NF-κB target genes distinct from those activated by classical proinflammatory mediators.
Regulation of Endothelial Cell Motility by Complexes of Tetraspan Molecules CD81/TAPA-1 and CD151/PETA-3 with α3β1 Integrin Localized at Endothelial Lateral Junctions
Authors: Yáñez-Mó María , Alfranca Arántzazu , Cabañas Carlos , Marazuela Mónica , Tejedor Reyes , Angeles Ursa M. , Ashman Leonie K. , de Landázuri Manuel O. , Sánchez-Madrid Francisco
Published: July 2002

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell–cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell–cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with α3β1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for α3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.
Plasma Transglutaminase in Hypertrophic Chondrocytes: Expression and Cell-specific Intracellular Activation Produce Cell Death and Externalization
Authors: Nurminskaya Maria , Magee Cordula , Nurminsky Dmitry , Linsenmayer Thomas F.
Published: July 2002

We previously used subtractive hybridization to isolate cDNAs for genes upregulated in chick hypertrophic chondrocytes (Nurminskaya, M., and T.F. Linsenmayer. 1996. Dev. Dyn. 206:260–271). Certain of these showed homology with the “A” subunit of human plasma transglutaminase (factor XIIIA), a member of a family of enzymes that cross-link a variety of intracellular and matrix molecules. We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA. Northern and enzymatic analyses confirm that the molecule is upregulated in hypertrophic chondrocytes (as much as eightfold). The enzymatic analyses also show that appreciable transglutaminase activity in the hypertrophic zone becomes externalized into the extracellular matrix. This externalization most likely is effected by cell death and subsequent lysis—effected by the transglutaminase itself. When hypertrophic chondrocytes are transfected with a cDNA construct encoding the zymogen of factor XIIIA, the cells convert the translated protein to a lower molecular weight form, and they initiate cell death, become permeable to macromolecules and eventually undergo lysis. Non-hypertrophic cells transfected with the same construct do not show these degenerative changes. These results suggest that hypertrophic chondrocytes have a novel, tissue-specific cascade of mechanisms that upregulate the synthesis of plasma transglutaminase and activate its zymogen. This produces autocatalytic cell death, externalization of the enzyme, and presumably cross-linking of components within the hypertrophic matrix. These changes may in turn regulate the removal and/or calcification of this hypertrophic matrix, which are its ultimate fates.
The Small Leucine-Rich Repeat Proteoglycan Biglycan Binds to α-Dystroglycan and Is Upregulated in Dystrophic Muscle
Authors: Bowe Mark A. , Mendis Duane B. , Fallon Justin R.
Published: July 2002

The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface. The α-/β-dystroglycan subcomplex of the DAPC forms a critical link between the cytoskeleton and the extracellular matrix. A ligand blot overlay assay was used to search for novel dystroglycan binding partners in postsynaptic membranes from Torpedo electric organ. An ∼125-kD dystroglycan-binding polypeptide was purified and shown by peptide microsequencing to be the Torpedo ortholog of the small leucine-rich repeat chondroitin sulfate proteoglycan biglycan. Biglycan binding to α-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant α-dystroglycan. The biglycan binding site was mapped to the COOH-terminal third of α-dystroglycan. Glycosylation of α-dystroglycan is not necessary for this interaction, but binding is dependent upon the chondroitin sulfate side chains of biglycan. In muscle, biglycan is detected at both synaptic and nonsynaptic regions. Finally, biglycan expression is elevated in muscle from the dystrophic mdx mouse. These findings reveal a novel binding partner for α-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix. These results also raise the possibility of a role for biglycan in the pathogenesis, and perhaps the treatment, of muscular dystrophy.
Calcineurin-GATA-6 pathway is involved in smooth muscle–specific transcription
Authors: Wada Hiromichi , Hasegawa Koji , Morimoto Tatsuya , Kakita Tsuyoshi , Yanazume Tetsuhiko , Abe Mitsuru , Sasayama Shigetake
Published: July 2002

Intracellular calcium is one of the important signals that initiates the myogenic program. The calcium-activated phosphatase calcineurin is necessary for the nuclear import of the nuclear factor of activated T cell (NFAT) family members, which interact with zinc finger GATA transcription factors. Whereas GATA-6 plays a role in the maintenance of the differentiated phenotype in vascular smooth muscle cells (VSMCs), it is unknown whether the calcineurin pathway is associated with GATA-6 and plays a role in the differentiation of VSMCs. The smooth muscle–myosin heavy chain (Sm-MHC) gene is a downstream target of GATA-6, and provides a highly specific marker for differentiated VSMCs. Using immunoprecipitation Western blotting, we showed that NFATc1 interacted with GATA-6. Consistent with this, NFATc1 further potentiated GATA-6–activated Sm-MHC transcription. Induction of VSMCs to the quiescent phenotype caused nuclear translocation of NFATc1. In differentiated VSMCs, blockage of calcineurin down-regulated the amount of GATA-6-DNA binding as well as the expression of Sm-MHC and its transcriptional activity. These findings demonstrate that the calcineurin pathway is associated with GATA-6 and is required for the maintenance of the differentiated phenotype in VSMCs.
α-Glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis
Authors: Gillmor C. Stewart , Poindexter Patricia , Lorieau Justin , Palcic Monica M. , Somerville Chris
Published: July 2002

Novel mutations in the RSW1 and KNOPF genes were identified in a large-scale screen for mutations that affect cell expansion in early Arabidopsis embryos. Embryos from both types of mutants were radially swollen with greatly reduced levels of crystalline cellulose, the principal structural component of the cell wall. Because RSW1 was previously shown to encode a catalytic subunit of cellulose synthase, the similar morphology of knf and rsw1-2 embryos suggests that the radially swollen phenotype of knf mutants is largely due to their cellulose deficiency. Map-based cloning of the KNF gene and enzyme assays of knf embryos demonstrated that KNF encodes α-glucosidase I, the enzyme that catalyzes the first step in N-linked glycan processing. The strongly reduced cellulose content of knf mutants indicates that N-linked glycans are required for cellulose biosynthesis. Because cellulose synthase catalytic subunits do not appear to be N glycosylated, the N-glycan requirement apparently resides in other component(s) of the cellulose synthase machinery. Remarkably, cellular processes other than extracellular matrix biosynthesis and the formation of protein storage vacuoles appear unaffected in knf embryos. Thus in Arabidopsis cells, like yeast, N-glycan trimming is apparently required for the function of only a small subset of N-glycoproteins.
Molecular Mechanisms of Lymphocyte Homing to Peripheral Lymph Nodes
Authors: Warnock R. Aaron , Askari Sanaz , Butcher Eugene C. , Andrian Ulrich H. von
Published: July 2002

To characterize the adhesion cascade that directs lymphocyte homing to peripheral lymph nodes (PLNs), we investigated the molecular mechanisms of lymphocyte interactions with the microvasculature of subiliac lymph nodes. We found that endogenous white blood cells and adoptively transferred lymph node lymphocytes (LNCs) tethered and rolled in postcapillary high endothelial venules (HEVs) and to a lesser extent in collecting venules. Similarly, firm arrest occurred nearly exclusively in the paracortical HEVs. Endogenous polymorphonuclear (PMNs) and mononuclear leukocytes (MNLs) attached and rolled in HEVs at similar frequencies, but only MNLs arrested suggesting that the events downstream of primary rolling interactions critically determine the specificity of lymphocyte recruitment. Antibody inhibition studies revealed that L-selectin was responsible for attachment and rolling of LNCs, and that LFA-1 was essential for sticking. LFA-1–dependent arrest was also abolished by pertussis toxin, implicating a requirement for Gαi−-protein–linked signaling. α4 integrins, which play a critical role in lymphocyte homing to Peyer's Patches, made no significant contribution to attachment, rolling, or sticking in resting PLNs. Velocity analysis of interacting LNCs revealed no detectable contribution by LFA-1 to rolling. Taken together, our results suggest that lymphocyte– HEV interactions within PLNs are almost exclusively initiated by L-selectin followed by a G protein–coupled lymphocyte-specific activation event and activation-induced engagement of LFA-1. These events constitute a unique adhesion cascade that dictates the specificity of lymphocyte homing to PLNs.
Cooperative Inhibition of  T-Cell Antigen Receptor Signaling by a Complex between a Kinase and a Phosphatase
Authors: Cloutier Jean-François , Veillette André
Published: July 2002

Antigen receptor–triggered T-cell activation is mediated by the sequential action of the Src and Syk/Zap-70 families of protein tyrosine kinases (PTKs). Previously, we reported that another PTK termed p50csk was a potent negative regulator of T-cell receptor (TCR) signaling because of its ability to inactivate Src-related kinases. This inhibitory effect required the catalytic activity of Csk, as well as its Src homology (SH)3 and SH2 domains. Subsequent studies uncovered that, via its SH3 domain, p50csk was associated with PEP, a proline-enriched protein tyrosine phosphatase (PTP) of unknown function expressed in hemopoietic cells. Herein, we have attempted to identify the role of the Csk-PEP complex in T lymphocytes. The results of our experiments showed that, like Csk, PEP was a strong repressor of TCR signaling. This property was dependent on the phosphatase activity of PEP, as well as on the sequence mediating its binding to p50csk. Through reconstitution experiments in Cos-1 cells, evidence was obtained that Csk and PEP act synergistically to inhibit protein tyrosine phosphorylation by Src-related kinases, and that this effect requires their association. Finally, experiments with a substrate-trapping mutant of PEP suggested that PEP functions by dephosphorylating and inactivating the PTKs responsible for T-cell activation. In addition to giving novel insights into the mechanisms involved in the negative regulation of T-cell activation, these findings indicate that the association of an inhibitory PTK with a PTP constitutes a more efficient means of inhibiting signal transduction by Src family kinases in vivo.