bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of a role for cyclic ADP-ribose in long-term synaptic depression in hippocampus
Authors: Reyes-Harde Magali , Empson Ruth , Potter Barry V. L. , Galione Antony , Stanton Patric K.
Published: July 2002

Ca
2+
released from presynaptic and postsynaptic intracellular stores plays important roles in activity-dependent synaptic plasticity, including long-term depression (LTD) of synaptic strength. At Schaffer collateral–CA1 synapses in the hippocampus, presynaptic ryanodine receptor-gated stores appear to mobilize some of the Ca
2+
necessary to induce LTD. Cyclic ADP-ribose (cADPR) has recently been proposed as an endogenous activator of ryanodine receptors in sea urchin eggs and several mammalian cell types. Here, we provide evidence that cADPR-mediated signaling pathways play a key role in inducing LTD. We show that biochemical production of cGMP increases cADPR concentration in hippocampal slices
in vitro
, and that blockade of cGMP-dependent protein kinase, cADPR receptors, or ryanodine-sensitive Ca
2+
stores each prevent the induction of LTD at Schaffer collateral–CA1 synapses. A lack of effect of postsynaptic infusion of either cADPR antagonist indicates a probable presynaptic site of action.
Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors
Authors: Polo John M. , Belli Barbara A. , Driver David A. , Frolov Ilya , Sherrill Scott , Hariharan Mangala J. , Townsend Kay , Perri Silvia , Mento Steven J. , Jolly Douglas J. , Chang Stephen M. W. , Schlesinger Sondra , Dubensky Thomas W.
Published: July 2002

Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10
7
infectious units/ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors.
Defense by foot adhesion in a beetle (
Hemisphaerota cyanea
)
Authors: Eisner Thomas , Aneshansley Daniel J.
Published: July 2002

The beetle
Hemisphaerota cyanea
(Chrysomelidae; Cassidinae) responds to disturbance by activating a tarsal adhesion mechanism by which it secures a hold on the substrate. Its tarsi are oversized and collectively bear some 60,000 adhesive bristles, each with two terminal pads. While walking, the beetle commits but a small fraction of the bristles to contact with the substrate. But when assaulted, it presses its tarsi flatly down, thereby touching ground with all or nearly all of the bristles. Once so adhered, it can withstand pulling forces of up to 0.8 g (≈60 times its body mass) for 2 min, and of higher magnitudes, up to >3 g, for shorter periods. Adhesion is secured by a liquid, most probably an oil. By adhering, the beetle is able to thwart attacking ants, given that it is able to cling more persistently than the ant persists in its assault. One predator, the reduviid
Arilus cristatus
, is able to feed on the beetle, possibly because by injecting venom it prevents the beetle from maintaining its tarsal hold.
Temporal dynamics of cortical representation for action
Authors: Nishitani N. , Hari R.
Published: July 2002

Brain-imaging studies have shown that the human Broca's region and precentral motor cortex are activated both during execution of hand actions and during observation of similar actions performed by other individuals. We aimed to clarify the temporal dynamics of this cortical activation by neuromagnetic recordings during execution, on-line imitation, and observation of right-hand reaching movements that ended with a precision pinch of the tip of a manipulandum. During execution, the left inferior frontal cortex [Brodmann's area (BA) 44] was activated first (peak ≈250 ms before the pinching); this activation was followed within 100–200 ms by activation in the left primary motor area (BA4) and 150–250 ms later in the right BA4. During imitation and observation, the sequence was otherwise similar, but it started from the left occipital cortex (BA19). Activation was always strongest during action imitation. Only the occipital activation was detected when the subject observed the experimenter reaching his hand without pinching. These results suggest that the left BA44 is the orchestrator of the human “mirror neuron system” and is strongly involved in action imitation. The mirror system matches action observation and execution and probably contributes to our understanding of actions made by others.
Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor α can be modulated by anti-tumor necrosis factor α therapy
Authors: Li Y. Y. , Feng Y. Q. , Kadokami T. , McTiernan C. F. , Draviam R. , Watkins S. C. , Feldman A. M.
Published: July 2002

Myocardial fibrosis caused by maladaptive extracellular matrix
(ECM) remodeling is implicated in the dysfunction of the failing heart.
Matrix metalloproteinases (MMPs) regulate ECM remodeling, and
are regulated by cytokines. Transgenic mice with cardiac-specific
overexpression of tumor necrosis factor α (TNF-α) (TNF1.6) develop
heart failure. We hypothesized that modulation of TNF-α and/or MMP
activity might alter the myocardial ECM remodeling process and the
development of heart failure. To test this hypothesis, we took
advantage of the TNF1.6 mice and studied soluble and total collagens
and collagen type profiling by using hydroxyproline quantification,
Sircol collagen assay, Northern blot analysis, and immunohistochemistry
and studied myocardial function by using echocardiography. Progressive
ventricular hypertrophy and dilation in the TNF1.6 mice were
accompanied by a significant increase in MMP-2 and MMP-9 activity, an
increase in collagen synthesis, deposition, and denaturation, and a
decrease in undenatured collagens. In young TNF1.6 mice, these changes
in the ECM were associated with marked diastolic dysfunction as
demonstrated by significantly reduced transmitral Doppler
echocardiographic E/A wave ratio. Anti-TNF-α treatment with
adenoviral vector expressing soluble TNF-α receptor type I attenuated
both MMP-2 and MMP-9 activity, prevented further collagen synthesis,
deposition and denaturation, and preserved myocardial diastolic
function in young, but not old, TNF1.6 mice. The results suggest a
critical role of TNF-α and MMPs in myocardial matrix remodeling and
functional regulation and support the hypothesis that both TNF-α and
MMPs may serve as potential therapeutic targets in the treatment of
heart failure.
Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases
Authors: Yang Meng , Baranov Eugene , Jiang Ping , Sun Fang-Xian , Li Xiao-Ming , Li Lingna , Hasegawa Satoshi , Bouvet Michael , Al-Tuwaijri Maraya , Chishima Takashi , Shimada Hiroshi , Moossa A. R. , Penman Sheldon , Hoffman Robert M.
Published: July 2002

We have imaged, in real time, fluorescent tumors growing and metastasizing in live mice. The whole-body optical imaging system is external and noninvasive. It affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals. We have established new human and rodent tumors that stably express very high levels of the
Aequorea victoria
green fluorescent protein (GFP) and transplanted these to appropriate animals. B16F0-GFP mouse melanoma cells were injected into the tail vein or portal vein of 6-week-old C57BL/6 and nude mice. Whole-body optical images showed metastatic lesions in the brain, liver, and bone of B16F0-GFP that were used for real time, quantitative measurement of tumor growth in each of these organs. The AC3488-GFP human colon cancer was surgically implanted orthotopically into nude mice. Whole-body optical images showed, in real time, growth of the primary colon tumor and its metastatic lesions in the liver and skeleton. Imaging was with either a trans-illuminated epifluorescence microscope or a fluorescence light box and thermoelectrically cooled color charge-coupled device camera. The depth to which metastasis and micrometastasis could be imaged depended on their size. A 60-μm diameter tumor was detectable at a depth of 0.5 mm whereas a 1,800-μm tumor could be visualized at 2.2-mm depth. The simple, noninvasive, and highly selective imaging of growing tumors, made possible by strong GFP fluorescence, enables the detailed imaging of tumor growth and metastasis formation. This should facilitate studies of modulators of cancer growth including inhibition by potential chemotherapeutic agents.
Different functional domains of TAF
II
250 modulate expression of distinct subsets of mammalian genes
Authors: O'Brien Thomas , Tjian Robert
Published: July 2002

The TATA box-binding protein-associated factors (TAFs) are thought to play an essential role in eukaryotic RNA polymerase II transcription by mediating the expression of distinct subsets of genes. In hamster ts13 cells, a single amino acid change in TAF
II
250, which disrupts its acetyl-transferase activity at the restrictive temperature, alters the transcription of specific genes involved in cell cycle control. Likewise, disruption of the amino-terminal kinase domain of TAF
II
250 results in transcriptional defects in ts13 cells. However, it was not known whether the acetyl-transferase or kinase domains of TAF
II
250 modulate specific classes of genes and whether these two domains regulate distinct subsets of genes. Here we have used high-density gene-profiling to identify mammalian transcripts that require either the TAF
II
250 acetyl-transferase or protein kinase function for proper expression. We found that transcription of at least 18% of genes are differentially expressed at the restrictive temperature. The promoter region of one of these genes was subsequently characterized, and both upstream elements as well as the core promoter were shown to be TAF
II
250 responsive. We also found that expression of ≈6% of genes in ts13 cells requires a functional TAF
II
250 amino-terminal kinase domain, but only ≈1% of these hamster genes also require the TAF
II
250 acetyl-transferase activity. Our results suggest that the two TAF
II
250 enzymatic activities are important for regulating largely nonoverlapping sets of genes involved in a wide range of biological functions
in vivo
.
Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase
Authors: Boyer Paul L. , Sarafianos Stefan G. , Arnold Edward , Hughes Stephen H.
Published: July 2002

We have examined amino acid substitutions at residues 115 and 116 in the reverse transcriptase (RT) of HIV-1. A number of properties were examined, including polymerization and processivity on both DNA and RNA templates, strand displacement, ribonucleotide misincorporation, and resistance to nucleoside analogs. The RT variants Tyr-115–Phe and Phe-116–Tyr are similar to wild-type HIV-1 RT in most, but not all, respects. In contrast, the RT variant Tyr-115–Val is significantly impaired in polymerase activity compared with wild-type RT; however, Tyr-115–Val is able to incorporate ribonucleotides as well as deoxyribonucleotides during polymerization and is resistant to a variety of nucleoside analogs.
The Vepsians: An Administratively Divided Nationality
Authors: Kurs Ott
Published: July 2002

The administrative division of late imperial Russia made few concessions to minority populations, who often found themselves divided among several provinces. The Bolshevik ascendancy to power changed the situation; Vladimir Lenin's “federal compromise” marked a breakthrough from the tsarist unitary practice to a system of governance which, at least on paper, made allowance for the ethnocultural diversity of the population. The chief designers of the Bolshevik nationality policy believed that a federal arrangement would offer a framework for controlling undesirable national sentiments during the transitional stage when class identities would gradually replace ethnic attachments. However, it turned out that for non-Russian groups the national-territorial autonomous units were not simply empty containers, free of cultural and emotional meaning, in which their political socialization would occur. These units became an integral part of their national identity; ethnicity obtained “legal” territorial roots and the various territorial units began to function as vessels of ethnic consciousness.
Human eosinophil–airway smooth muscle cell interactions
Authors: Hughes J. Margaret , Arthur Craig A. , Baracho Shane , Carlin Stephen M. , Hawker Kristen M. , Johnson Peter R.A. , Armour Carol L.
Published: July 2002

Eosinophils are present throughout the airway wall of asthmatics. The nature of the interaction between human airway smooth muscle cells (ASMC) and eosinophils was investigated in this study. We demonstrated, using light microscopy, that freshly isolated eosinophils from healthy donors rapidly attach to ASMC in vitro. Numbers of attached eosinophils were highest at 2 h, falling to 50% of maximum by 20 h. Eosinophil attachment at 2 h was reduced to 72% of control by anti‐VCAM‐1, and to 74% at 20 h by anti‐ICAM‐1. Pre‐treatment of ASMC for 24 h with TNF‐α, 10 nM, significantly increased eosinophil adhesion to 149 and 157% of control after 2 and 20 h. These results provide evidence that eosinophil interactions with ASMC involve VCAM‐1 and ICAM‐1 and are modulated by TNF‐α.
Investigations of the relationship betw een disease and airborne (1→3)‐β‐D‐glucan in buildings
Authors: Rylander Ragnar
Published: July 2002

Studies on the relationship between symptoms in indoor air and the amount of airborne (1→3)‐β‐D‐glucan were reviewed. Relationships were found for symptoms and objective tests of airways inflammation. The data suggest that (1→3)‐β‐D‐glucan could be a causative agent.
Lipoxygenase-Mediated N-Demethylation of Imipramine and Related Tricyclic Antidepressants in the Presence of Hydrogen Peroxide
Authors: Hu Jianan , Sajan Mini , Kulkarni Arun P.
Published: July 2002

In this study, we examined the ability of soybean lipoxygenase to mediate the N-demethylation of imipramine and related drugs in the presence of hydrogen peroxide. Formaldehyde generation resulting from the N-demethylation of imipramine, a prototype drug, was found to depend on incubation time, and the concentration of the enzyme, imipramine, and hydrogen peroxide. Under optimal assay conditions, Vmax values of 14 to 18 nmol formaldehyde/min/nmol enzyme or 133 to 164 nmol formaldehyde/min/mg protein were observed. An inhibition of formaldehyde and desipramine formation by nordihydroguaiaretic acid confirmed the lipoxygenase involvement. The blockade of the reaction by glutathione, dithiothreitol, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) indicated the generation of a free radical intermediate from imipramine. Desipramine, trimipramine, clomipramine, and diltiazem, but not amitriptyline and doxepin, were also oxidized, albeit at a lower rate. Collectively, the evidence gathered in this study suggests, for the first time, that tricyclic antidepressant drugs may undergo lipoxygenase-catalyzed N-demethylation.
Inhibited Cytotoxic Leukocyte Activity in Tilapia (Oreochromis niloticus) Following Exposure to Immunotoxic Chemicals
Authors: Smith D. A. , Schurig G. G. , Smith S. A. , Holladay S. D.
Published: July 2002

The measure of the ability of cytotoxic immune cells to target and lyse foreign cells has been widely used as a predictor of im-munosuppression in chemical-exposed rodents. However, the efficacy of this function for predicting immunosuppressive chemical exposure in nonrodent species remains unknown. In the present report, tilapia ( Oreochromis niloticus) were exposed to 9 chemical agents known to inhibit rodent cytotoxic T lymphocyte (CTL) activity in mice, benzo[a]pyrene (B[a]P), 7, 12-dimethylbenzanthracene (DMBA), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), dimethyl-nitrosamine (DMN), cadmium chloride (CdCl2), azathioprine (AZA), T2 mycotoxin (T2 toxin), hexachlorocyclohexane (lindane), and diethylstilbesterol(DES); and five chemical agents which do not inhibit this response, oxymethalone, acetonitrile, tert-butylhydro-quinone (TBHQ), toluene, and formaldehyde. Eight of the nine agents which inhibit rodent CTL responses also caused decreased cytolytic responses in fish. All five of the compounds with negative CTL effects in rodents were also negative in fish. Thus, 13 of the 14 chemical agents tested gave similar results in fish as reported in rodents, indicating a comparable pattern of inhibited immune cell cytolytic responses in chemical-exposed tilapia to that seen in the laboratory rodent models.
The Regulatory Perspective on the Role of Toxicology in Protecting the Public Health
Authors: Green Sidney
Published: July 2002

The role of toxicology as far as regulatory agencies are concerned is to assist in determining the safety of products/substances to which humans and wildlife are exposed. This is accomplished primarily by assessing the adverse effects of these products/substances. In a number of cases, the benefits of the product/substance are balanced against the adverse effects or hazards. Often the stimulus for new guidelines, policies, and regulations is the discovery of a heretofore unknown hazard, or the presumption that a hazard exists for which current methodologies are deemed insufficient or totally lacking. An example of the latter type of stimulus are the so-called, endocrine modulators or disruptors. Research in toxicology also serves as a stimulus for new guidelines, policies and regulations, for example, dietary restriction and transgenic animal models. There is a painstaking process associated with the development of policy by regulatory agencies regardless of which stimulus operates. This process, however, does not meet with success in all instances. One of the major difficulties in establishing new guidelines, policies, and regulations, is that of bringing about a balance between risk and safety. The role of toxicology in providing basic information which is then used to make decisions and bring about this balance is pivotal. Toxicology can be the stimulus for new guidelines, policies, and regulations, i.e., contribute early in the process or it can contribute at a later stage in providing information to substantiate or refute the need for new guidelines, policies, or regulations.
Toxicity of Tetryl (N-Methyl-N,2,4,6–Tetranitroaniline) in F344 Rats
Authors: Reddy Tirumuru V. , Olson Greg R. , Wiechman Barry , Reddy Gunda , Torsella Joni , Daniel F. Bernard , Leach Glenn J.
Published: July 2002

The toxicity of tetryl (N-methyl-N,2,4,6–tetranitroaniline) in male and female F344 rats was evaluated after adminstration in the diet for 14 or 90 days. The 14–day study diet concentrations used were 0, 500, 1250, 2000, 2500, and 5000 ppm; the 90–day study diet concentrations were 0, 200, 1000, and 3000 ppm tetryl in the diet. The calculated average daily tetryl intake was 32.1, 82.5, 130.3, 178.9, and 374.4 mg/kg body weight (BW) for females and 31.8, 80.0, 121.0, 170.5, and 349.7 mg/kg BW for males in the 14–day study. For the 90–day studies, the daily tetryl intake was 14.2, 68.8, and 199.0 mg/kg BW for females and 13.0, 62.4, and 179.6 mg/kg BW for males. In the 14–day study, there was a significant decrease in body weights (males), whereas relative(organ/body weight) liver and spleen (females), and kidney (males) weights were significantly increased in the 5000–ppm dose group. Hematological effects observed were decreased hemoglobin and hematocrit and an increased number of reticulocyts in females (2000 to 5000 ppm). Methemoglobin levels in males (2000 to 5000 ppm) and females (5000 ppm) and total blood protein and albumin levels in all groups of males and females (except 500 ppm) were significantly increased. Histopathological changes were observed in kidneys (deposition of cytoplasmic droplets) of all dose groups of male rats. In the sub–chronic (90–day) study, feed intake was reduced in all dose groups, but a significant decrease in terminal body weights was observed in females (1000 and 3000 ppm) and males (3000 ppm). An increase in the relative liver, kidney (1000–3000 ppm), and spleen (3000 ppm) weights were noted in both sexes. The hemoglobin content and red blood cell count were decreased whereas the reticulocyte count was elevated (3000 ppm) in both sexes at 45 and 90 days. Methemoglobin levels were increased in both sexes (1000 and 3000 ppm). Histopathologicalchanges were noted in the spleen (pigment deposition and erythroid cell hyperplasia) of both sexes (3000 ppm) and kidneys (tubular degeneration and cytoplasmic droplets containing alpha-2-micro globulin) of male rats (1000 to 3000 ppm). A no observed adverse effect level (NOAEL) for both sexes was 13 mg/kg BW/day was determined.
Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion
Authors: Chitaev Nikolai A. , Troyanovsky Sergey M.
Published: July 2002

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion.
The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho
Authors: Leeuwen Frank N. van , Kain Hendrie E.T. , Kammen Rob A. van der , Michiels Frits , Kranenburg Onno W. , Collard John G.
Published: July 2002

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.
Multistep Navigation and the Combinatorial Control of Leukocyte Chemotaxis
Authors: Foxman Ellen F. , Campbell James J. , Butcher Eugene C.
Published: July 2002

Cells migrating within tissues may encounter multiple chemoattractant signals in complex spatial and temporal patterns. To understand leukocyte navigation in such settings, we have explored the migratory behavior of neutrophils in model scenarios where they are presented with two chemoattractant sources in various configurations. We show that, over a wide range of conditions, neutrophils can migrate “down” a local chemoattractant gradient in response to a distant gradient of a different chemoattractant. Furthermore, cells can chemotax effectively to a secondary distant agonist after migrating up a primary gradient into a saturating, nonorienting concentration of an initial attractant. Together, these observations suggest the potential for cells' step-by-step navigation from one gradient to another in complex chemoattractant fields. The importance of such sequential navigation is confirmed here in a model system in which neutrophil homing to a defined domain (a) requires serial responses to agonists presented in a defined spatial array, and (b) is a function of both the agonist combination and the sequence in which gradients are encountered. We propose a multistep model of chemoattractant-directed migration, which requires that leukocytes display multiple chemoattractant receptors for successful homing and provides for combinatorial determination of microenvironmental localization.
Two Distinct Pathways for Targeting Proteins from the Cytoplasm to the Vacuole/Lysosome
Authors: Baba Misuzu , Osumi Masako , Scott Sidney V. , Klionsky Daniel J. , Ohsumi Yoshinori
Published: July 2002

Stress conditions lead to a variety of physiological responses at the cellular level. Autophagy is an essential process used by animal, plant, and fungal cells that allows for both recycling of macromolecular constituents under conditions of nutrient limitation and remodeling the intracellular structure for cell differentiation. To elucidate the molecular basis of autophagic protein transport to the vacuole/lysosome, we have undertaken a morphological and biochemical analysis of this pathway in yeast.

Using the vacuolar hydrolase aminopeptidase I (API) as a marker, we provide evidence that the autophagic pathway overlaps with the biosynthetic pathway, cytoplasm to vacuole targeting (Cvt), used for API import. Before targeting, the precursor form of API is localized mostly in restricted regions of the cytosol as a complex with spherical particles (termed Cvt complex). During vegetative growth, the Cvt complex is selectively wrapped by a membrane sac forming a double membrane-bound structure of ∼150 nm diam, which then fuses with the vacuolar membrane. This process is topologically the same as macroautophagy induced under starvation conditions in yeast (Baba, M., K. Takeshige, N. Baba, and Y. Ohsumi. 1994. J. Cell Biol. 124:903–913). However, in contrast with autophagy, API import proceeds constitutively in growing conditions. This is the first demonstration of the use of an autophagy-like mechanism for biosynthetic delivery of a vacuolar hydrolase. Another important finding is that when cells are subjected to starvation conditions, the Cvt complex is now taken up by an autophagosome that is much larger and contains other cytosolic components; depending on environmental conditions, the cell uses an alternate pathway to sequester the Cvt complex and selectively deliver API to the vacuole. Together these results indicate that two related but distinct autophagy-like processes are involved in both biogenesis of vacuolar resident proteins and sequestration of substrates to be degraded.
Neurofilament-dependent Radial Growth of Motor Axons and Axonal Organization of Neurofilaments Does Not Require the Neurofilament Heavy Subunit (NF-H) or Its Phosphorylation
Authors: Rao Mala V. , Houseweart Megan K. , Williamson Toni L. , Crawford Thomas O. , Folmer Janet , Cleveland Don W.
Published: July 2002

Neurofilaments are essential for establishment and maintenance of axonal diameter of large myelinated axons, a property that determines the velocity of electrical signal conduction. One prominent model for how neurofilaments specify axonal growth is that the 660–amino acid, heavily phosphorylated tail domain of neurofilament heavy subunit (NF-H) is responsible for neurofilament-dependent structuring of axoplasm through intra-axonal crossbridging between adjacent neurofilaments or to other axonal structures. To test such a role, homologous recombination was used to generate NF-H–null mice. In peripheral motor and sensory axons, absence of NF-H does not significantly affect the number of neurofilaments or axonal elongation or targeting, but it does affect the efficiency of survival of motor and sensory axons. Loss of NF-H caused only a slight reduction in nearest neighbor spacing of neurofilaments and did not affect neurofilament distribution in either large- or small-diameter motor axons. Since postnatal growth of motor axon caliber continues largely unabated in the absence of NF-H, neither interactions mediated by NF-H nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.
Sli15 Associates with the Ipl1 Protein Kinase to Promote Proper Chromosome Segregation in Saccharomyces cerevisiae
Authors: Kim Jae-hyun , Kang Jung-seog , Chan Clarence S.M.
Published: July 2002

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. We show here that sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole–associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation.
Ezrin Promotes Morphogenesis of Apical Microvilli and Basal Infoldings in Retinal Pigment Epithelium
Authors: Bonilha Vera Lúcia , Finnemann Silvia C. , Rodriguez-Boulan Enrique
Published: July 2002

Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infoldings typical of these cells, and characterize the role of ezrin in these processes using antisense and transfection approaches. In the adult rat RPE, only ezrin (no moesin or radixin) was detected at high levels by immunofluorescence and immunoelectron microscopy at microvilli and basal infoldings. At the time when these morphological differentiations develop, in the first two weeks after birth, ezrin levels increased fourfold to adult levels. Addition of ezrin antisense oligonucleotides to primary cultures of rat RPE drastically decreased both apical microvilli and basal infoldings. Transfection of ezrin cDNA into the RPE-J cell line, which has only trace amounts of ezrin and moesin, sparse and stubby apical microvilli, and no basal infoldings, induced maturation of microvilli and the formation of basal infoldings without changing moesin expression levels. Taken together, the results indicate that ezrin is a major determinant in the maturation of surface differentiations of RPE independently of other ERM family members.
Role of Egr1 in Hippocampal Synaptic Enhancement Induced by Tetanic Stimulation and Amputation
Authors: Wei Feng , Xu Zao C. , Qu Zhican , Milbrandt Jeffrey , Zhuo Min
Published: July 2002

Hippocampal neurons fire spikes when an animal is at a particular location or performs certain behaviors in a particular place, providing a cellular basis for hippocampal involvement in spatial learning and memory. In a natural environment, spatial memory is often associated with potentially dangerous sensory experiences such as noxious or painful stimuli. The central sites for such pain-associated memory or plasticity have not been identified. Here we present evidence that excitatory glutamatergic synapses within the CA1 region of the hippocampus may play a role in storing pain-related information. Peripheral noxious stimulation induced excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal cells in anesthetized animals. Tissue/nerve injury caused a rapid increase in the level of the immediate-early gene product Egr1 (also called NGFI-A, Krox24, or zif/268) in hippocampal CA1 neurons. In parallel, synaptic potentiation induced by a single tetanic stimulation (100 Hz for 1 s) was enhanced after the injury. This enhancement of synaptic potentiation was absent in mice lacking Egr1. Our data suggest that Egr1 may act as an important regulator of pain-related synaptic plasticity within the hippocampus.
p38 Map Kinase Mediates Bax Translocation in Nitric Oxide–Induced Apoptosis in Neurons
Authors: Ghatan Saadi , Larner Stephen , Kinoshita Yoshito , Hetman Michal , Patel Leena , Xia Zhengui , Youle Richard J. , Morrison Richard S.
Published: July 2002

Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP) kinase in neurons. In the present study, we determined if NO-induced cell death in neurons was dependent on p38 MAP kinase activity. Sodium nitroprusside (SNP), an NO donor, elevated caspase activity and induced death in human SH-SY5Y neuroblastoma cells and primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminished caspase induction and protected SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor zVAD-fmk did not provide significant protection. A role for p38 MAP kinase was further substantiated by the observation that SB203580 blocked translocation of the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct activator of p38 MAP kinase promoted Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons were resistant to SNP, further demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway.
Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network
Authors: Wilcke Mona , Johannes Ludger , Galli Thierry , Mayau Véronique , Goud Bruno , Salamero Jean
Published: July 2002

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.
Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter
Authors: Müller Waltraud G. , Walker Dawn , Hager Gordon L. , McNally James G.
Published: July 2002

We have examined the relationship between transcription and chromatin structure using a tandem array of the mouse mammary tumor virus (MMTV) promoter driving a ras reporter. The array was visualized as a distinctive fluorescent structure in live cells stably transformed with a green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR), which localizes to the repeated MMTV elements after steroid hormone treatment. Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2). Within 3 h after hormone addition, arrays visualized by GFP-GR or DNA fluorescent in situ hybridization (FISH) decondensed to varying degrees, in the most pronounced cases from a ∼0.5-μm spot to form a fiber 1–10 μm long. Arrays later recondensed by 3–8 h of hormone treatment. The degree of decondensation was proportional to the amount of transcript produced by the array as detected by RNA FISH. Decondensation was blocked by two different drugs that inhibit polymerase II, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) and α-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin. They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously.
A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice
Authors: Wehling Michelle , Spencer Melissa J. , Tidball James G.
Published: July 2002

Dystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy.
Role of heparan sulfate as a tissue-specific regulator of FGF-4 and FGF receptor recognition
Authors: Allen Benjamin L. , Filla Mark S. , Rapraeger Alan C.
Published: July 2002

FGF signaling uses receptor tyrosine kinases that form high-affinity complexes with FGFs and heparan sulfate (HS) proteoglycans at the cell surface. It is hypothesized that assembly of these complexes requires simultaneous recognition of distinct sulfation patterns within the HS chain by FGF and the FGF receptor (FR), suggesting that tissue-specific HS synthesis may regulate FGF signaling. To address this, FGF-2 and FGF-4, and extracellular domain constructs of FR1-IIIc (FR1c) and FR2-IIIc (FR2c), were used to probe for tissue-specific HS in embryonic day 18 mouse embryos. Whereas FGF-2 binds HS ubiquitously, FGF-4 exhibits a restricted pattern, failing to bind HS in the heart and blood vessels and failing to activate signaling in mouse aortic endothelial cells. This suggests that FGF-4 seeks a specific HS sulfation pattern, distinct from that of FGF-2, which is not expressed in most vascular tissues. Additionally, whereas FR2c binds all FGF-4–HS complexes, FR1c fails to bind FGF-4–HS in most tissues, as well as in Raji-S1 cells expressing syndecan-1. Proliferation assays using BaF3 cells expressing either FR1c or FR2c support these results. This suggests that FGF and FR recognition of specific HS sulfation patterns is critical for the activation of FGF signaling, and that synthesis of these patterns is regulated during embryonic development.
Requirement of the juxtamembrane domain of the cadherin cytoplasmic tail for morphogenetic cell rearrangement during myotome development
Authors: Horikawa Kazuki , Takeichi Masatoshi
Published: July 2002

During development, the activity of cadherin cell adhesion molecules is assumed to be regulated to allow for cell rearrangement or translocation. Previous studies suggest that the juxtamembrane (JM) domain of the cadherin cytoplasmic tail, which contains the site for binding to p120ctn, has a regulatory function in this adhesion system. To study the possible role of JM domain–dependent cadherin regulation in embryonic cell rearrangement, we ectopically expressed a series of N-cadherin mutants in developing somites of chicken embryos. When a JM domain–deficient N-cadherin was expressed, the morphogenetic expansion of the myotome was strongly suppressed. However, a triple alanine substitution in the JM domain, which specifically inhibited the p120ctn binding, had no effect on myotome development. Furthermore, a dominant negative N-cadherin, which had a deletion at the extracellular domain but maintained the normal cytoplasmic tail, did not affect myotome expansion; although it disrupted intersomite boundaries. Overexpression of p120ctn also did not affect myotome expansion, but it did perturb myofiber orientation. These and other observations suggest that the JM domain of N-cadherin has a regulatory role in myotome cell rearrangement in which molecules other than p120ctn are involved. The p120ctn molecule itself seems to play a critical role in the arrangement of myofibers.
In vivo binding of active heat shock transcription factor 1 to human chromosome 9 heterochromatin during stress
Authors: Jolly Caroline , Konecny Lara , Grady Deborah L. , Kutskova Yulia A. , Cotto José J. , Morimoto Richard I. , Vourc'h Claire
Published: July 2002

Activation of the mammalian heat shock transcription factor (HSF)1 by stress is a multistep process resulting in the transcription of heat shock genes. Coincident with these events is the rapid and reversible redistribution of HSF1 to discrete nuclear structures termed HSF1 granules, whose function is still unknown. Key features are that the number of granules correlates with cell ploidy, suggesting the existence of a chromosomal target. Here we show that in humans, HSF1 granules localize to the 9q11-q12 heterochromatic region. Within this locus, HSF1 binds through direct DNA–protein interaction with a nucleosome-containing subclass of satellite III repeats. HSF1 granule formation only requires the DNA binding competence and the trimerization of the factor. This is the first example of a transcriptional activator that accumulates transiently and reversibly on a chromosome-specific heterochromatic locus.
Conditional disruption of β1 integrin in Schwann cells impedes interactions with axons
Authors: Feltri M. Laura , Porta Diana Graus , Previtali Stefano C. , Nodari Alessandro , Migliavacca Barbara , Cassetti Arianna , Littlewood-Evans Amanda , Reichardt Louis F. , Messing Albee , Quattrini Angelo , Mueller Ulrich , Wrabetz Lawrence
Published: July 2002

In dystrophic mice, a model of merosin-deficient congenital muscular dystrophy, laminin-2 mutations produce peripheral nerve dysmyelination and render Schwann cells unable to sort bundles of axons. The laminin receptor and the mechanism through which dysmyelination and impaired sorting occur are unknown. We describe mice in which Schwann cell–specific disruption of β1 integrin, a component of laminin receptors, causes a severe neuropathy with impaired radial sorting of axons. β1-null Schwann cells populate nerves, proliferate, and survive normally, but do not extend or maintain normal processes around axons. Interestingly, some Schwann cells surpass this problem to form normal myelin, possibly due to the presence of other laminin receptors such as dystroglycan and α6β4 integrin. These data suggest that β1 integrin links laminin in the basal lamina to the cytoskeleton in order for Schwann cells to ensheath axons, and alteration of this linkage contributes to the peripheral neuropathy of congenital muscular dystrophy.