bluuebunny/crossref_metadata_2025 huggingface dataset:
Resistance to excitotoxin-induced seizures and neuronal death in mice lacking the preprotachykinin A gene
Authors: Liu Hantao , Cao Yuqing , Basbaum Allan I. , Mazarati Andrey M. , Sankar Raman , Wasterlain Claude G.
Published: July 2002

Epileptic seizures are associated with increases in hippocampal excitability, but the mechanisms that render the hippocampus hyperexcitable chronically (in epilepsy) or acutely (in status epilepticus) are poorly understood. Recent evidence suggests that substance P (SP), a peptide that has been implicated in cardiovascular function, inflammatory responses, and nociception
,
also contributes to hippocampal excitability and status epilepticus, in part by enhancing glutamate release. Here we report that mice with disruption of the preprotachykinin A gene, which encodes SP and neurokinin A, are resistant to kainate excitoxicity. The mice show a reduction in the duration and severity of seizures induced by kainate or pentylenetetrazole, and both necrosis and apoptosis of hippocampal neurons are prevented. Although kainate induced the expression of bax and caspase 3 in the hippocampus of wild-type mice, these critical intracellular mediators of cell death pathways were not altered by kainate injection in the mutant mice. These results indicate that the reduction of seizure activity and the neuroprotection observed in preprotachykinin A null mice are caused by the extinction of a SP/neurokinin A-mediated signaling pathway that is activated by seizures. They suggest that these neurokinins are critical to the control of hippocampal excitability, hippocampal seizures, and hippocampal vulnerability.
Feeding behavior in dopamine-deficient mice
Authors: Szczypka Mark S. , Rainey Mark A. , Kim Douglas S. , Alaynick William A. , Marck Brett T. , Matsumoto Alvin M. , Palmiter Richard D.
Published: July 2002

Mice that cannot make dopamine (DA), a condition caused by the selective inactivation of tyrosine hydroxylase in dopaminergic neurons, are born normal but gradually become hypoactive and hypophagic, and die at 3 weeks of age. We characterized the feeding and locomotor responses of these DA-deficient (DA−/−) mice to 3,4-dihyroxy-
l
-phenylalanine (
l
-DOPA) to investigate the relationship between brain DA levels and these complex behaviors. Daily administration of
l
-DOPA to DA−/− mice stimulated locomotor activity that lasted 6 to 9 hr; during that time the mice consumed most of their daily food and water. The minimal dose of
l
-DOPA that was sufficient to elicit normal feeding behavior in the DA−/− mice also restored their striatal DA to 9.1% of that in the wild-type (WT) mice at 3 hr; then DA content declined to <1% of WT levels by 24 hr. This dose of
l
-DOPA induced locomotor activity that exceeded that of treated WT mice by 5- to 7-fold, suggesting that DA−/− mice are supersensitive to DA. Unexpectedly, DA−/− mice manifested a second wave of activity 24 to 48 hr after
l
-DOPA treatment that was equivalent in magnitude to that of WT mice and independent of DA receptor activation. The DA−/− mice approached, sniffed, and chewed food during this second period of activity, but they ate <10% of that required for sustenance. Therefore, DA−/− mice can execute behaviors necessary to seek and ingest food, but they do not eat enough to survive.
Ectopic expression of E47 or E12 promotes the death of E2A-deficient lymphomas
Authors: Engel Isaac , Murre Cornelis
Published: July 2002

Mice with null mutations in the E2A gene are highly susceptible to the spontaneous development of thymic lymphomas. To understand better how E2A deficiency may contribute to lymphomagenesis, we have observed the consequences of enforced expression of the E2A gene products E12 and E47 in cell lines derived from lymphomas that arose spontaneously in E2A-deficient mice. E2A-expressing cells are steadily eliminated from lymphoma cultures into which E47 or E12 was introduced. The mechanism underlying the loss of E2A-expressing cells does not involve an arrest in cell-cycle progression. Rather, the E2A proteins activate a programmed cell death pathway in these lymphomas. This E2A-mediated cell death appears to be preceded by a loss of mitochondrial transmembrane potential. These data provide direct evidence that E2A gene products can act as tumor suppressors.
Recombination and transcription of the endogenous Ig heavy chain locus is effected by the Ig heavy chain intronic enhancer core region in the absence of the matrix attachment regions
Authors: Sakai Eiko , Bottaro Andrea , Davidson Laurie , Sleckman Barry P. , Alt Frederick W.
Published: July 2002

The intronic Ig heavy chain (IgH) enhancer, which consists of the core enhancer flanked by 5′ and 3′ matrix attachment regions, has been implicated in control of IgH locus recombination and transcription. To elucidate the regulatory functions of the core enhancer and its associated matrix attachment regions in the endogenous IgH locus, we have introduced targeted deletions of these elements, both individually and in combination, into an IgHa/b-heterozygous embryonic stem cell line. These embryonic stem cells were used to generate chimeric mice by recombination activating gene-2 (Rag-2)-deficient blastocyst complementation, and the effects of the introduced mutations were assayed in mutant B cells. We find that the core enhancer is necessary and sufficient to promote normal variable (V), diversity (D), and joining (J) segment recombination in developing B lineage cells and IgH locus transcription in mature B cells. Surprisingly, the 5′ and 3′ matrix attachment regions were dispensable for these processes.
Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids
Authors: Kochs Georg , Haller Otto
Published: July 2002

Interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases. It exhibits antiviral activity against a variety of RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Here, we report that MxA blocks the transport of Thogoto virus nucleocapsids into the nucleus, thereby preventing transcription of the viral genome. This interaction can be abolished by a mAb that neutralizes the antiviral activity of MxA. Our results reveal an antiviral mechanism whereby an interferon-induced protein traps the incoming virus and interferes with proper transport of the viral genome to its ultimate target compartment within the infected cell.
Hormonal prevention of breast cancer: Mimicking the protective effect of pregnancy
Authors: Guzman Raphael C. , Yang Jason , Rajkumar Lakshmanaswamy , Thordarson Gudmundur , Chen Xiaoyan , Nandi Satyabrata
Published: July 2002

Full term pregnancy early in life is the most effective natural protection against breast cancer in women. Rats treated with chemical carcinogen are similarly protected by a previous pregnancy from mammary carcinogenesis. Proliferation and differentiation of the mammary gland does not explain this phenomenon, as shown by the relative ineffectiveness of perphenazine, a potent mitogenic and differentiating agent. Here, we show that short term treatment of nulliparous rats with pregnancy levels of estradiol 17β and progesterone has high efficacy in protecting them from chemical carcinogen induced mammary cancers. Because the mammary gland is exposed to the highest physiological concentrations of estradiol and progesterone during full term pregnancy, it is these elevated levels of hormones that likely induce protection from mammary cancer. Thus, it appears possible to mimic the protective effects of pregnancy against breast cancer in nulliparous rats by short term specific hormonal intervention.
CD4+T cell-mediated control of a γ-herpesvirus in B cell-deficient mice is mediated by IFN-γ
Authors: Christensen Jan P. , Cardin Rhonda D. , Branum Kristen C. , Doherty Peter C.
Published: July 2002

The lack of B cells and antibody does not prevent mice from dealing effectively with a pathogenic γ-herpesvirus. Both CD4+and CD8+T cells contribute to the control of virus replication in the respiratory tract, with the depletion of either lymphocyte subset leading to increased titers in the lung. However, the further neutralization of IFN-γ diminishes the effectiveness of the CD4+T cell response and causes substantially increased mortality. Experiments with bone marrow radiation chimeras indicate that the immune CD4+effectors operate optimally when there is the potential for direct interaction with virus-infected targets expressing MHC class II glycoproteins, suggesting that the IFN-γ produced by these lymphocytes is functioning at short range. The numbers of latently infected cells in the spleens of carrier mice are also significantly increased by the concurrent depletion of both the CD4+population and IFN-γ. These experiments raise the possibility that the defective control of intercurrent γ-herpesvirus infections in patients with AIDS not only is due solely to the absence of helper T cells but also reflects the loss of an important set of CD4+effectors.
AIS
is an oncogene amplified in squamous cell carcinoma
Authors: Hibi Kenji , Trink Barry , Patturajan Meera , Westra William H. , Caballero Otávia L. , Hill David E. , Ratovitski Edward A. , Jen Jin , Sidransky David
Published: July 2002

We and others recently isolated a human
p53
homologue (
p40
/
p51
/
p63
/
p73L
) and localized the gene to the distal long arm of chromosome 3. Here we sought to examine the role of
p40
/
p73L
, two variants lacking the N-terminal transactivation domain, in cancer. Fluorescent
in situ
hybridization (FISH) analysis revealed frequent amplification of this gene locus in primary squamous cell carcinoma of the lung and head and neck cancer cell lines. (We named this locus
AIS
for
a
mplified
i
n
s
quamous cell carcinoma.) Furthermore, amplification of the
AIS
locus was accompanied by RNA and protein overexpression of a variant p68
AIS
lacking the terminal transactivation domain. Protein overexpression in primary lung tumors was limited to squamous cell carcinoma and tumors known to harbor a high frequency of
p53
mutations. Overexpression of p40
AIS
in Rat 1a cells led to an increase in soft agar growth and tumor size in mice. Our results support the idea that
AIS
plays an oncogenic role in human cancer.
Architecture with designer atoms: Simple theoretical considerations
Authors: Remacle F. , Levine R. D.
Published: July 2002

The distinct electronic states of assemblies of metallic quantum
dots are discussed in a simple approximation where each dot is mimicked
as an “atom” that carries one valence electron. Because of their
large size, the charging energy of the dots,
I
=
energy required to add another electron, is much smaller than for
ordinary atoms. The Coulomb blocking of charge migration is therefore
easier to overcome. For the theory, however, this is a challenge,
because ionic states, which are typically higher in energy, come down,
so the density of electronic states is high, and special methods need
to be adapted. Quantum dots are prepared by wet chemical methods and
accordingly are not quite identical. They will have a size distribution
that can be narrow (when the dots can be assembled into an ordered
array) or broad. Other sources of disorder are packing imperfections,
which are characteristic of a wider size distribution, ligand
deformations, and chemical unevenness. Two experimental control
parameters are the size of the dots and the spacing between them. We
discuss the combined effects of the low charging energy and disorder
and examine the distinct electronic phases that can be realized.
Modifications of proteins by polyunsaturated fatty acid peroxidation products
Authors: Refsgaard Hanne H. F. , Tsai Lin , Stadtman Earl R.
Published: July 2002

The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O
2
] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.
BSE as an organizational construction: a case study on the globalization of risk
Authors: Tacke Veronika
Published: July 2002

This article examines the BSE problem as an example of the ‘globalization of risk’. In order to determine whether the ‘globalization of risk’ is a social construction depending on the context, the paper emphasizes the particular role of organizations. It makes an empirical comparison of the BSE‐related risk‐constructions of five business associations in the German meat industry sector. The results show that the associations construct the risk in close relation to their horizons of globalization, thereby reflecting provision problems, which the companies they are representing face. While the main organizational domains in the sector tried to cope with the risk problem by different means of local market ‘closure’, one association, founded in reaction to the BSE problem, took over a ‘reflexive’ role with regard to the emerging risk communication on BSE in Germany.
L'avenir de l'approche par élaboration de règles
Authors: Larivée Serge , Boulerice Bernard , Perrier François , La Rocque Gabriel
Published: July 2002

L'objectif de cette étude vise la mise au point d'un instrument diagnostique informatisé basé sur l'approche par élaboration de règles proposée par Siegler en vue d'évaluer l'acquisition du scheme de la quantification des probabilités chez des sujets de 6 à 18 ans. Trois versions du test ont été utilisées: la version 1, version originale de Siegler (quatre types de problèmes et quatre règles); la version 2, comportant cinq nouvelles règles et la version 3 comportant les cinq nouvelles règles et onze types de problèmes. Les résultats montrent que la version 1 ne permet de classer que 50% des sujets, la version 2, 60% et la version 3, 70%. Malgré les améliorations apportées à la procédure d'évaluation, des lacunes subsistent, dont la principale est l'impossibilité d'évaluer la variabilité intra‐individuelle. Conscient des limites de son approche, Siegler ne lui attribue qu'un statut de modèle local de résolution de problèmes et lui substitue un modèle développemental du traitement de l'information d'inspiration darwinienne de variation et de sélection des stratégies. Les auteurs posent l'hypothèse que l'ingénierie informatique pourrait non seulement redonner à l'approche par élaboration de régies son statut général, mais permettrait aussi la prise en considération de la variabilité intra‐individuelle.
The Albanians of Kosovo and Macedonia: Ethnic Identity Superseding Religion
Authors: Babuna Aydin
Published: July 2002

The Albanians are divided between three religions: the Catholics, the Orthodox, and the Muslims. The religious groups participated in the national development of the Albanians around 1900 each in its own way and in proportion to its own strength. As the majority group, the Muslims (70–80%) played the most important role. Most of the Muslims wanted to remain within the Ottoman Empire as long as their traditional rights were maintained, in spite of a strong desire for local self-government, but, as the Ottoman Empire declined, radical Albanian leaders stressing cultural and linguistic unity rather than religious unity were to gain the upper hand. The Albanian language, culture, and feeling of common blood played the most important roles in Albanian nationalism. The Albanian language in particular gave Albanians the feeling of belonging to the same nation.
The Mordvins: Dilemmas of Mobilization in a Biethnic Community
Authors: Iurchenkov Valerii
Published: July 2002

The Mordvins are the largest and the southernmost Finno-Ugrian nationality in Russia. Their titular autonomous unit, the Mordovian Republic, is located in the southern part of the Volga-Viatka economic region, on the border between the forest and forest-steppe zones. The ethnic composition of today's Mordovia is mixed. According to the 1989 census Russians constituted 60.8% of the population, Mordvins stood in second place with 32.5% and Tatars ranked third with 4.9%.
Raising School Performance in the League Tables (Part 1): Disentangling the effects of social disadvantage
Authors: Levaçić Rosalind , Woods Philip A.
Published: July 2002

Abstract The article uses data from a longitudinal study of over 300 secondary schools to investigate differences in the rate at which schools improved General Certificate of Secondary Education (GCSE) examination results over the period 1991‐98 and the reasons for such differences. Two variables found to have the most impact on examination improvement were a low concentration of social disadvantage relative to other local schools and starting from a low base level of GCSE results. Furthermore, schools with high concentrations of social disadvantage were liable to suffer a dual handicap as their relative social disadvantage tended to worsen over time. Schools with better examination improvement slightly increased their budget share per pupil over the period studied while those performing least well experienced a reduction in budget share per pupil.
The Manipulation and Measurement of Task-specific Memory Self-Efficacy in Younger and Older Adults
Authors: Gardiner Maria , Luszcz Mary A. , Bryan Janet
Published: July 2002

Task-specific memory self-efficacy (TSMSE) was experimentally manipulated through provision of information about task difficulty, to determine its effect on free recall for 56 older (age 63-86) and 56 younger (age 16-25) adults. The implications of using prediction-based measures of TSMSE were addressed. After completing one recall trial of a list of 20 words, half the participants were told a second list comprised more difficult words; the others were told the second list would be similar to the first they had received. Free recall and TSMSE were measured before and after this manipulation. The manipulation reduced TSMSE for participants expecting a harder list of words, but not differently for younger compared with older adults. Younger and older adults’ recall declined at the second recall trial, but there was no difference between those expecting a harder list and those expecting a similar list. Recall was predicted by domain-specific memory self-efficacy as well as a traditional measure of TSMSE. The study demonstrated the malleability of memory self-efficacy, but called into question assertions about its salience as a mediator of older adults’ poorer memory performance.
Studies on the biological effects of ozone: 8. Effects on the total antioxidant status and on interleukin‐8 production
Authors: Bocci V. , Valacchi G. , Corradeschi F. , Fanetti G.
Published: July 2002

Ozone (O3) is a controversial gas because, owing to its potent oxidant properties, it exerts damaging effects on the respiratory tract and yet it has been used for four decades as a therapy. While the disinfectant activity of O3 is understandable, it is less clear how other biological effects can be elicited in human blood with practically no toxicity. On the other hand plasma and cells are endowed with a powerful antioxidant system so that a fairly wide range of O3 concentrations between 40 and 80μ g/ml per gram of blood (˜0.83‐1.66 mM) are effective but not deleterious. After blood ozonation total antioxidant status (TAS) and plasma protein thiol groups (PTG) decrease by 20% and 25%, respectively, while thiobarbituric acid reactive substances (TBARS) increases up to fivefold. The increase of haemolysis is negligible suggesting that the erythrocyte membrane is spared at the expense of other sacrificial substrates. While there is a clear relationship between the ozone dose and IL‐8 levels, we have noticed that high TAS and PTG values inhibit the cytokine production. This is in line with the current idea that hydrogen peroxide, as a byproduct of O3 decomposition, acts as a messenger for the cytokine induction.
Predicting Treatment Response to Naltrexone: The Influence of Craving and Family History
Authors: Monterosso John R. , Flannery Barbara A. , Pettinati Helen M. , Oslin David W. , Rukstalis Margaret , O'Brien Charles P. , Volpicelli Joseph R.
Published: July 2002

Naltrexone has repeatedly been shown to reduce drinking in alcohol‐dependent patients. Previous clinical research suggests that naltrexone may be more effective at reducing drinking among patients with high levels of alcohol craving at the beginning of treatment. In addition, laboratory studies suggest that naltrexone may be more efficacious among patients with a high familial loading of alcohol problems. We explored both of these possibilities in the context of the first 12‐week phase of a double blind, placebo‐controlled naltrexone trial.

A total of 121 patients were randomized to receive 100 mg/day naltrexone and 62 patients were randomized to receive placebo. Both naltrexone and placebo were given in conjunction with a psychosocial intervention designed to be integrated with the use of pharmacotherapy. This intervention was administered by nurse practitioners.

Overall, patients randomized to naltrexone reported drinking five or more drinks on fewer days than did placebo controls (p = .04). Interactions were observed between medication group assignment and both craving level prior to randomization (p = .02) and family loading of alcohol problems (p = .05). In both cases, the interaction was in the predicted direction. These data suggest that patients with high levels of alcohol craving or a strong family history of alcoholism are more likely to benefit from naltrexone treatment.
Absence of Detectable Toxicity in Rats Fed Partially Hydrolyzed Guar Gum (K-13) for 13 Weeks
Authors: Koujitani Takatoshi , Oishi Hidetoshi , Kubo Yuji , Maeda Toshihiro , Sekiya Keiji , Yasuba Masashi , Matsuoka Nobuo , Nishimura Koichi
Published: July 2002

Toxicity studies were conducted to evaluate acute and subchronic oral toxicity and mutagenicity of partially hydrolyzed guar gum (K-13). In an acute toxicity study, mice and rats were treated with K-13 at a dose of 6000 mg/ kg. There were no deaths, so the LD50s were >6000 mg/ kg in both species. In a subchronic toxicity study, K-13 was given to rats as a dietary admixture at concentrations of 0.2. 1.0 and 5.0% for 13 weeks. There were no effects attributable to K-13 in any examinations. K-13 proved to have no mutagenic potential in a reverse mutation test using bacteria.
Desmin Is Essential for the Tensile Strength and Integrity of Myofibrils but Not for Myogenic Commitment, Differentiation, and Fusion of Skeletal Muscle
Authors: Li Zhenlin , Mericskay Mathias , Agbulut Onnik , Butler-Browne Gillian , Carlsson Lena , Thornell Lars-Eric , Babinet Charles , Paulin Denise
Published: July 2002

A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des −/−) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362–366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255– 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des −/− mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.
Major Histocompatibility Complex Class II Compartments in Human and Mouse B Lymphoblasts Represent Conventional Endocytic Compartments
Authors: Kleijmeer Monique J. , Morkowski Stanislaw , Griffith Janice M. , Rudensky Alexander Y. , Geuze Hans J.
Published: July 2002

In most human and mouse antigen-presenting cells, the majority of intracellular major histocompatibility complex (MHC) class II molecules resides in late endocytic MHC class II compartments (MIICs), thought to function in antigen processing and peptide loading. However, in mouse A20 B cells, early endocytic class II-containing vesicles (CIIVs) have been reported to contain most of the intracellular MHC class II molecules and have also been implicated in formation of MHC class II–peptide complexes. To address this discrepancy, we have studied in great detail the endocytic pathways of both a human (6H5.DM) and a mouse (A20.Ab) B cell line. Using quantitative immunoelectron microscopy on cryosections of cells that had been pulse–chased with transferrin-HRP or BSA-gold as endocytic tracers, we have identified up to six endocytic subcompartments including an early MIIC type enriched in invariant chain, suggesting that it serves as an important entrance to the endocytic pathway for newly synthesized MHC class II/invariant chain complexes. In addition, early MIICs represented the earliest endocytic compartment containing MHC class II– peptide complexes, as shown by using an antibody against an abundant endogenous class II–peptide complex. The early MIIC exhibited several though not all of the characteristics reported for the CIIV and was situated just downstream of early endosomes. We have not encountered any special class II-containing endocytic structures besides those normally present in nonantigen-presenting cells. Our results therefore suggest that B cells use conventional endocytic compartments rather than having developed a unique compartment to accomplish MHC class II presentation.
Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein
Authors: Sakai Norio , Sasaki Keiko , Ikegaki Natsu , Shirai Yasuhito , Ono Yoshitaka , Saito Naoaki
Published: July 2002

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.
Polymerization of Purified Yeast Septins: Evidence That Organized Filament Arrays May Not Be Required for Septin Function
Authors: Frazier Jennifer A. , Wong Mei Lie , Longtine Mark S. , Pringle John R. , Mann Matthias , Mitchison Timothy J. , Field Christine
Published: July 2002

The septins are a family of proteins required for cytokinesis in a number of eukaryotic cell types. In budding yeast, these proteins are thought to be the structural components of a filament system present at the mother–bud neck, called the neck filaments. In this study, we report the isolation of a protein complex containing the yeast septins Cdc3p, Cdc10p, Cdc11p, and Cdc12p that is capable of forming long filaments in vitro. To investigate the relationship between these filaments and the neck filaments, we purified septin complexes from cells deleted for CDC10 or CDC11. These complexes were not capable of the polymerization exhibited by wild-type preparations, and analysis of the neck region by electron microscopy revealed that the cdc10Δ and cdc11Δ cells did not contain detectable neck filaments. These results strengthen the hypothesis that the septins are the major structural components of the neck filaments. Surprisingly, we found that septin dependent processes like cytokinesis and the localization of Bud4p to the neck still occurred in cdc10Δ cells. This suggests that the septins may be able to function in the absence of normal polymerization and the formation of a higher order filament structure.
Nucleocytoplasmic Shuttling of the Cdc42p Exchange Factor Cdc24p
Authors: Nern Aljoscha , Arkowitz Robert A.
Published: July 2002

Cdc24p, the GDP/GTP exchange factor for the regulator of actin cytoskeleton Cdc42p, localizes to sites of polarized growth. Here we show that Cdc24p shuttles in and out of the yeast nucleus during vegetative growth. Far1p is necessary and sufficient for nuclear accumulation of Cdc24p, suggesting that its nuclear import occurs via an association with Far1p. Nuclear export is triggered either by entry into the cell cycle or by mating pheromone. As Far1p is degraded upon entry into the cell cycle, cell cycle–dependent export of Cdc24p occurs in the absence of Far1p, whereas during mating similar export kinetics indicate that a Cdc24p–Far1p complex is exported. Our results suggest that the nucleus serves as a store of preformed Cdc24p–Far1p complex which is required for chemotropism.
Activation by Cdc42 and Pip2 of Wiskott-Aldrich Syndrome Protein (Wasp) Stimulates Actin Nucleation by Arp2/3 Complex
Authors: Higgs Henry N. , Pollard Thomas D.
Published: July 2002

We purified native WASp (Wiskott-Aldrich Syndrome protein) from bovine thymus and studied its ability to stimulate actin nucleation by Arp2/3 complex. WASp alone is inactive in the presence or absence of 0.5 μM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate (PIP2) micelles allowed WASp to activate actin nucleation by Arp2/3 complex, and this was further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP2 and Cdc42 concentrated around lipid micelles and vesicles, providing that Cdc42 was GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 μM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction.
Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product
Authors: Tucker Karen E. , Berciano Maria Teresa , Jacobs Erica Y. , LePage David F. , Shpargel Karl B. , Rossire Jennifer J. , Chan Edward K.L. , Lafarga Miguel , Conlon Ronald A. , Matera A. Gregory
Published: July 2002

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.
Telomeres act autonomously in maize to organize the meiotic bouquet from a semipolarized chromosome orientation
Authors: Carlton Peter M. , Cande W. Zacheus
Published: July 2002

During meiosis, chromosomes undergo large-scale reorganization to allow pairing between homologues, which is necessary for recombination and segregation. In many organisms, pairing of homologous chromosomes is accompanied, and possibly facilitated, by the bouquet, the clustering of telomeres in a small region of the nuclear periphery. Taking advantage of the cytological accessibility of meiosis in maize, we have characterized the organization of centromeres and telomeres throughout meiotic prophase. Our results demonstrate that meiotic centromeres are polarized prior to the bouquet stage, but that this polarization does not contribute to bouquet formation. By examining telocentric and ring chromosomes, we have tested the cis-acting requirements for participation in the bouquet. We find that: (a) the healed ends of broken chromosomes, which contain telomere repeats, can enter the bouquet; (b) ring chromosomes enter the bouquet, indicating that terminal position on a chromosome is not necessary for telomere sequences to localize to the bouquet; and (c) beginning at zygotene, the behavior of telomeres is dominant over any centromere-mediated chromosome behavior. The results of this study indicate that specific chromosome regions are acted upon to determine the organization of meiotic chromosomes, enabling the bouquet to form despite large-scale changes in chromosome architecture.
Vaccination with DNA Encoding the Immunodominant LACK Parasite Antigen Confers Protective Immunity to Mice Infected with Leishmania major
Authors: Gurunathan Sanjay , Sacks David L. , Brown Daniel R. , Reiner Steven L. , Charest Hughes , Glaichenhaus Nicolas , Seder Robert A.
Published: July 2002

To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-γ (IFN-γ) production. Moreover, both the enhancement of IFN-γ production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8+ T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8+ T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.
Essential Lymphocyte Function Associated 1 (LFA-1): Intercellular Adhesion Molecule Interactions for T Cell–mediated B Cell Apoptosis by Fas/APO-1/CD95
Authors: Wang Jin , Lenardo Michael J.
Published: July 2002

B cells are susceptible to Fas ligand (FasL)+ CD4+ Th1 cell–mediated apoptosis. We demonstrate that blocking the interactions between lymphocyte function associated (LFA)-1 and intercellular adhesion molecule(ICAM)-1 and ICAM-2 completely suppresses Fas-dependent B cell lysis. Antibodies to CD2 and CD48 partially suppress B cell apoptosis, whereas anti-B7.1 and anti-B7.2 antibodies have no effect. Also, B cells from ICAM-1–deficient mice are resistant to FasL+ T cell–mediated death. Our results suggest that LFA-1/ICAM interactions are crucial for Th1 cell–mediated B cell apoptosis and may contribute to the maintenance of B cell homeostasis in vivo.
The Coordinated Action of CC Chemokines in the Lung Orchestrates Allergic Inflammation and Airway Hyperresponsiveness
Authors: Gonzalo Jose-Angel , Lloyd Clare M. , Wen Danyi , Albar Juan P. , Wells Timothy N.C. , Proudfoot Amanda , Martinez-A C. , Dorf Martin , Bjerke Torbjörn , Coyle Anthony J. , Gutierrez-Ramos Jose-Carlos
Published: July 2002

The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness (BHR) that characterize asthma is achieved by the regulated accumulation and activation of different leukocyte subsets in the lung. The development and maintenance of these processes correlate with the coordinated production of chemokines. Here, we have assessed the role that different chemokines play in lung allergic inflammation and BHR by blocking their activities in vivo. Our results show that blockage of each one of these chemokines reduces both lung leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES (regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1α, reduces only slightly lung eosinophilia and BHR. Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators. These results suggest that different chemokines activate different cellular and molecular pathways that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their individual blockage results in intervention at different levels of these processes.
Ribp, a Novel Rlk/Txk- and Itk-Binding Adaptor Protein That Regulates T Cell Activation
Authors: Rajagopal Keshava , Sommers Connie L. , Decker Donna C. , Mitchell Elizabeth O. , Korthauer Ulf , Sperling Anne I. , Kozak Christine A. , Love Paul E. , Bluestone Jeffrey A.
Published: July 2002

A novel T cell–specific adaptor protein, RIBP, was identified based on its ability to bind Rlk/Txk in a yeast two-hybrid screen of a mouse T cell lymphoma library. RIBP was also found to interact with a related member of the Tec family of tyrosine kinases, Itk. Expression of RIBP is restricted to T and natural killer cells and is upregulated substantially after T cell activation. RIBP-disrupted knockout mice displayed apparently normal T cell development. However, proliferation of RIBP-deficient T cells in response to T cell receptor (TCR)-mediated activation was significantly impaired. Furthermore, these activated T cells were defective in the production of interleukin (IL)-2 and interferon γ, but not IL-4. These data suggest that RIBP plays an important role in TCR-mediated signal transduction pathways and that its binding to Itk and Rlk/Txk may regulate T cell differentiation.
Gata-3 Induces T Helper Cell Type 2 (Th2) Cytokine Expression and Chromatin Remodeling in Committed Th1 Cells
Authors: Lee Hyun Jun , Takemoto Naofumi , Kurata Hirokazu , Kamogawa Yumiko , Miyatake Shoichiro , O'Garra Anne , Arai Naoko
Published: July 2002

Committed T helper type 1 (Th1) and Th2 effector cells, resulting from chronic antigenic stimulation in interleukin (IL)-12 and IL-4, are implicated in the pathology of autoimmune and allergic diseases. Committed Th1 cells cannot be induced to change their cytokine profiles in response to antigenic stimulation and Th2 cytokine–inducing conditions. Here, we report that ectopic expression of GATA-3 induced Th2-specific cytokine expression not only in developing Th1 cells but also in otherwise irreversibly committed Th1 cells and a Th1 clone, HDK1. Moreover, cAMP, an inhibitor of cytokine production by Th1 cells, markedly augmented Th2 cytokine production in GATA-3–expressing Th1 cells. Ectopic expression of GATA-3 in developing Th1 cells, but not in Th1 clone HDK1, induced endogenous GATA-3, suggesting an autoregulatory mechanism for maintenance of GATA-3 expression in Th2 cells. Structure–function analyses of GATA-3 revealed that the NH2-terminal transactivation domain and the COOH-terminal zinc finger domain of GATA-3 were critical, whereas the NH2-terminal zinc finger domain was dispensable for the induction of IL-4. Both zinc fingers, however, were required for IL-5 induction. A Th2-specific DNaseI-hypersensitive site of the IL-4 locus was detected in GATA-3–expressing Th1 cells. Thus, GATA-3 can change the phenotype of committed Th1 cells, previously considered to be irreversible.
Thiol-Mediated Redox Regulation of Intestinal Lamina Propria T Lymphocytes
Authors: Sido Bernd , Braunstein Jutta , Breitkreutz Raoul , Herfarth Christian , Meuer Stefan C.
Published: July 2002

Intestinal lamina propria T lymphocytes (LP-Ts) have a markedly low proliferative potential both in vivo and in vitro. Here, we have identified that the capacity of antigen-presenting cells to release cysteine upon receptor–ligand interactions represents a critical parameter for proliferation of LP-Ts. The availability of cysteine is limiting for the intracellular production of glutathione, which in turn is essential for cell cycle progression. When cysteine is provided either directly or by addition of the reducing agent 2-mercaptoethanol to cystine-containing culture medium, proliferation of LP-T is fully restored. Importantly, coculture with peripheral blood monocytes that easily take up cystine, reduce cystine, and secrete cysteine also restores reactivity of LP-Ts to T cell receptor/CD3 stimulation. In marked contrast, lamina propria macrophages lack this capacity to elaborate cysteine, and thereby secure physiological unresponsiveness to antigen exposure in the intestinal microenvironment. The well-documented local recruitment of blood monocytes in inflammatory bowel disease (IBD) may thus represent an important parameter underlying hyperresponsiveness of T cells, an essential component of the pathogenesis of IBD.
Requirements for Bone Marrow–Derived Antigen-Presenting Cells in Priming Cytotoxic T Cell Responses to Intracellular Pathogens
Authors: Lenz Laurel L. , Butz Eric A. , Bevan Michael J.
Published: July 2002

Bone marrow (BM)-derived antigen-presenting cells (APCs) are potent stimulators of T cell immune responses. We investigated the requirements for antigen presentation by these cells in priming cytotoxic T lymphocyte (CTL) responses to intracellular bacterial and viral pathogens. [Parent→F1] radiation BM chimeras were constructed using C57BL/6 donors and (C57BL/6 × BALB/c)F1 recipients. Infection of chimeric mice with either Listeria monocytogenes or vaccinia virus expressing the nucleoprotein (NP) antigen from lymphocytic choriomeningitis virus (LCMV) primed H2-Db–restricted, but not H2-Kd–restricted CTL responses, demonstrating the requirement for BM-derived APCs for successful priming of CTL responses to these pathogens. Surprisingly, this did not hold true for chimeric mice infected with LCMV itself. LCMV-infected animals developed strong CTL responses specific for both H2-Db– and H2-Ld–restricted NP epitopes. These findings indicate that in vivo priming of CTL responses to LCMV is remarkably insensitive to deficiencies in antigen presentation by professional BM-derived APCs.