bluuebunny/crossref_metadata_2025 huggingface dataset:
Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein
Authors: Burton Melissa , Nakai Hiroyuki , Colosi Peter , Cunningham Janet , Mitchell Rachel , Couto Linda
Published: July 2002

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1α promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL/6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2–10 μg/ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200–400 ng/ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.
Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1β converting enzyme cDNA
Authors: Oshikawa Katsuhisa , Shi Fushun , Rakhmilevich Alexander L. , Sondel Paul M. , Mahvi David M. , Yang Ning-Sun
Published: July 2002

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1β converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-γ. First, we compared the levels of IFN-γ secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-γ production from splenocytesin vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-γ levels were measured at the skin transfection sitein vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression.In vivodepletion experiments showed that this antitumor effect was CD8+T cell-mediated and partially IFN-γ-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8+T cells and IFN-γ-dependent pathways.
A cyclodextrin dimer with a photocleavable linker as a possible carrier for the photosensitizer in photodynamic tumor therapy
Authors: Ruebner Anja , Yang Zhiwei , Leung David , Breslow Ronald
Published: July 2002

A cyclodextrin dimer has been synthesized with two β-cyclodextrins linked by a flexible chain containing a carbon–carbon double bond. This dimer binds and solubilizes a phthalocyanine-based photosensitizer that generates singlet oxygen on irradiation. When the complex is irradiated, the singlet oxygen cleaves the carbon–carbon link, and the cyclodextrins are released, liberating the photosensitizer into the light path. Ideas about how this phenomenon could be used to make photodynamic tumor therapy into a more selective process are described.
Multiple and diverse forms of regulated exocytosis in wild-type and defective PC12 cells
Authors: Kasai Haruo , Kishimoto Takuya , Liu Ting-Ting , Miyashita Yasushi , Podini Paola , Grohovaz Fabio , Meldolesi Jacopo
Published: July 2002

Regulated exocytosis triggered by the photolysis of a caged Ca
2+
compound, DM-nitrophen, was investigated by patch-clamp capacitance measurements in two clones of PC12, the first wild-type and the second (PC12-27) defective of both types of classical secretory vesicles together with the neuronal-type receptors for the attachment proteins of the N-ethylmaleimide-sensitive fusion protein, the so called SNAREs. Moreover, the electrophysiological data were correlated with the ultrastructure of resting quick-frozen–freeze-dried cells of the two clones. Wild-type PC12 exhibited two-component capacitance responses, time constants of 30–100 ms and >10 s, that previous studies had suggested to reflect primarily the fusion of the small and large secretory vesicles, each contributing cell surface increases of ≈10%. Both of these components were largely and specifically inhibited whether cells previously were microinjected with tetanus toxin light chain. In the defective clone, large responses also were recorded (≈19% surface expansion; time constant, ≈1 s) that, in contrast to those of the wild-type, were entirely resistant to the toxin. Although secretory organelles, i.e., large vesicles and also profiles of small vesicles, were abundant at the cell periphery and often docked to the plasmalemma of resting wild-type PC12, in the defective clone, no superficial accumulation of vesicles was observed. Our coordinate structural and functional results have revealed diversities between the two classical forms of regulated secretion in wild-type PC12 and have provided evidence of a toxin-insensitive form of Ca
2+
-induced exocytosis, prominent in the defective clone, that may play an important role(s) in cellular physiology.
Regulation of selectin binding activity by cyclization of sialic acid moiety of carbohydrate ligands on human leukocytes
Authors: Mitsuoka Chikako , Ohmori Katsuyuki , Kimura Naoko , Kanamori Akiko , Komba Shiro , Ishida Hideharu , Kiso Makoto , Kannagi Reiji
Published: July 2002

We provide here evidence that supports the occurrence of a biologically dormant form of selectin ligand carbohydrate, the sialyl 6-sulfo Lewis X containing modified sialic acid, in human leukocytes. The modification of sialic acid involves first de-N-acetylation of sialic acid moiety through ubiquitous de-N-acetylation/re-N-acetylation cycle, followed by the dehydrative cyclization of de-N-acetyl sialic acid to form “cyclic sialic acid.” The enzyme involved in the dehydration of de-N-acetyl sialic acid is a calcium-dependent enzyme having neutral–alkaline pH optimum. De-N-acetyl sialyl 6-sulfo Lewis X retained selectin binding activity as well as parental sialyl 6-sulfo Lewis X, but cyclic sialyl 6-sulfo Lewis X was devoid of selectin binding activity. Sialyl 6-sulfo Lewis X carrying the cyclic sialic acid is specifically recognized by the newly generated mAb, G159. The determinant was distributed widely among normal human leukocytes, especially on monocytes and subsets of lymphocytes including NK cells, helper memory T cells, Tcr-γδ T cells, and a part of B cells. The determinant was detected also on several cultured lymphocytic leukemia cell lines andO-tetradecanoylphorbol 13-acetate-activated lymphoid cells. Cyclic sialyl 6-sulfo Lewis X is efficiently formed by the action of the partly membrane-bound calcium-dependent enzyme, tentatively called “sialic acid cyclase,” and a possible physiological significance of this reaction could be a rapid inactivation of selectin binding activity at the cell surface. Conversely, the accumulated intracellular cyclic sialyl 6-sulfo Lewis X determinant may function as a dormant pool of selectin ligands, which, on appropriate stimulation, is hydrolyzed and becomes active in selectin-dependent cell adhesion.
Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV
Authors: Spitsin S. , Steplewski K. , Fleysh N. , Belanger H. , Mikheeva T. , Shivprasad S. , Dawson W. , Koprowski H. , Yusibov V.
Published: July 2002

Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in
Nicotiana benthamiana
,
Nicotiana tabacum
MD609, and
Spinacia oleracea
. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.
Reconstitution of functional L-selectin ligands on a cultured human endothelial cell line by cotransfection of α1→3 fucosyltransferase VII and newly cloned GlcNAcβ:6-sulfotransferase cDNA
Authors: Kimura Naoko , Mitsuoka Chikako , Kanamori Akiko , Hiraiwa Nozomu , Uchimura Kenji , Muramatsu Takashi , Tamatani Takuya , Kansas Geoffrey S. , Kannagi Reiji
Published: July 2002

Recently, we proposed sialyl 6-sulfo Lewis X as a major carbohydrate-capping group of the L-selectin ligands on high endothelial venules in human lymph nodes. In this study we succeeded in reconstituting functional L-selectin ligands on a cultured human endothelial cell line, ECV304, by transfecting the α1→3fucosyltranseferase VII (Fuc-T VII) and newly cloned GlcNAcβ:6-sulfotransferase (6-Sul-T) cDNAs. The ECV304 cells transfected with Fuc-T VII cDNA expressed conventional sialyl Lewis X detected with specific antibodies including 2H5, whereas the cells transfected with 6-Sul-T cDNA expressed sialyl 6-sulfo lactosamine as well as MECA-79-defined carbohydrate determinants, but these singly transfected cells failed to express sialyl 6-sulfo Lewis X, as detected with the antisialyl 6-sulfo Lewis X mAb G152. Sialyl 6-sulfo Lewis X appeared only on the cells that were cotransfected with both 6-Sul-T and Fuc-T VII cDNAs. Significant adhesion of L-selectin-expressing cells was seen only to the doubly transfected ECV304 cells and was inhibited by G152. No adhesion was observed to the cells transfected either with 6-Sul-T or with Fuc-T VII cDNA alone. The mRNAs of the two enzymes were expressed or were inducible upon interleukin 1 stimulation in human endothelial cells. These results indicate that a set of carbohydrate determinants synthesized by the concerted action of the two enzymes, as typically represented by the sialyl 6-sulfo Lewis X-capping group, serves as an essential component of the ligand for L-selectin and that the reagents 2H5 and MECA-79, utilized in earlier studies to detect L-selectin ligand on high endothelial venules, recognize two different aspects of the same set of synthetic products.
T cell factor-activated transcription is not sufficient to induce anchorage-independent growth of epithelial cells expressing mutant β-catenin
Authors: Barth Angela I. M. , Stewart Daniel B. , Nelson W. James
Published: July 2002

N-terminal mutations in β-catenin that inhibit β-catenin degradation are found in primary tumors and cancer cell lines, and increased β-catenin/T cell factor (TCF)-activated transcription in these cells has been correlated with cancer formation. However, the role of mutant β-catenin in cell transformation is poorly understood. Here, we compare the ability of different N-terminal mutations of β-catenin (ΔN131, ΔN90, ΔGSK) to induce TCF-activated transcription and anchorage-independent growth in Madin–Darby canine kidney epithelial cells. Expression of ΔN90 or ΔGSK β-catenin increased TCF-activated transcription but did not induce significant anchorage-independent cell growth. In contrast, deletion of the α-catenin-binding site in ΔN131 β-catenin reduced TCF-activated transcription, compared with that induced by ΔN90 or ΔGSK β-catenin, but significantly enhanced anchorage-independent cell growth.
A complex of iron and nucleic acid catabolites is a signal that triggers differentiation in a freshwater protozoan
Authors: Smith-Somerville Harriett E. , Hardman John K. , Timkovich Russell , Ray William J. , Rose Karen E. , Ryals Phillip E. , Gibbons Sandra H. , Buhse Howard E.
Published: July 2002

The polymorphic ciliated protozoan
Tetrahymena vorax
can undergo differentiation from the microstomal form, which normally feeds on bacteria and other particulate matter, into the macrostomal cell type, which is capable of ingesting prey ciliates. The process is triggered by exposure of the microstome to an inducer contained in stomatin, an exudate of the prey. To establish the identity of the signal, stomatin was fractionated by combinations of cation exchange, HPLC, and TLC, and the fractions were assayed for biological activity. Although no single active fraction of purified inducer was obtained, all fractions with activity contained ferrous iron and the nucleic acid catabolites hypoxanthine (6-oxypurine) and uracil (2,4-dioxopyrimidine), probably in a chelated form. The activity of synthetic complexes containing these three components is equivalent to stomatin. These results indicate a role for ferrous iron and its potential in chelated form to signal differentiation in certain protozoa and, perhaps, in other organisms as well.
Pentameric assembly of a neuronal glutamate transporter
Authors: Eskandari Sepehr , Kreman Michael , Kavanaugh Michael P. , Wright Ernest M. , Zampighi Guido A.
Published: July 2002

Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in
Xenopus
laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 ± 5 nm
2
) predicted 35 ± 3 transmembrane α-helices in the transporter complex. This information along with secondary structure models (6–10 transmembrane α-helices) suggested an oligomeric state for EAAT3. EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110°. Each domain appeared to contribute to an extracellular mass that projects ≈3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.
Seed plant phylogeny inferred from all three plant genomes: Monophyly of extant gymnosperms and origin of Gnetales from conifers
Authors: Chaw Shu-Miaw , Parkinson Christopher L. , Cheng Yuchang , Vincent Thomas M. , Palmer Jeffrey D.
Published: July 2002

Phylogenetic relationships among the five groups of extant seed plants are presently quite unclear. For example, morphological studies consistently identify the Gnetales as the extant sister group to angiosperms (the so-called “anthophyte” hypothesis), whereas a number of molecular studies recover gymnosperm monophyly, and few agree with the morphology-based placement of Gnetales. To better resolve these and other unsettled issues, we have generated a new molecular data set of mitochondrial small subunit rRNA sequences, and have analyzed these data together with comparable data sets for the nuclear small subunit rRNA gene and the chloroplast
rbcL
gene. All nuclear analyses strongly ally Gnetales with a monophyletic conifers, whereas all mitochondrial analyses and those chloroplast analyses that take into account saturation of third-codon position transitions actually place Gnetales
within
conifers, as the sister group to the Pinaceae. Combined analyses of all three genes strongly support this latter relationship, which to our knowledge has never been suggested before. The combined analyses also strongly support monophyly of extant gymnosperms, with cycads identified as the basal-most group of gymnosperms,
Ginkgo
as the next basal, and all conifers except for Pinaceae as sister to the Gnetales + Pinaceae clade. According to these findings, the Gnetales may be viewed as extremely divergent conifers, and the many morphological similarities between angiosperms and Gnetales (e.g., double fertilization and flower-like reproductive structures) arose independently.
Fertile and diploid nuclear transplants derived from embryonic cells of a small laboratory fish, medaka (
Oryzias latipes
)
Authors: Wakamatsu Yuko , Ju Bensheng , Pristyaznhyuk Inna , Niwa Katsutoshi , Ladygina Tatiana , Kinoshita Masato , Araki Kazuo , Ozato Kenjiro
Published: July 2002

Fertile and diploid nuclear transplants were successfully generated
by using embryonic cells as donors in a small laboratory fish, medaka
(
Oryzias latipes
). Embryonic cell nuclei from transgenic
fish carrying the green fluorescent protein (
GFP
) gene
were transplanted into unfertilized eggs enucleated by x-ray
irradiation. In this study, 1 out of 588 eggs transplanted in the first
experiment and 5 out of 298 eggs transplanted in the second experiment
reached the adult stage. All of these nuclear transplants were fertile
and diploid, and the natural and GFP markers of the donor nuclei were
transmitted to the F
1
and F
2
offspring in a
Mendelian fashion. This systematic study proves the feasibility of
generating nuclear transplants by using embryonic cells from fish as
donors, and it is supported by convincing evidence.
Copper-64-diacetyl-bis(
N
4
-methylthiosemicarbazone): An agent for radiotherapy
Authors: Lewis Jason S. , Laforest Richard , Buettner Thomas L. , Song Sheng-Kwei , Fujibayashi Yasuhisa , Connett Judith M. , Welch Michael J.
Published: July 2002

Systemic administration of hypoxia-selective
64
Cu-diacetyl-bis(
N
4
-methylthiosemicarbazone)
(
64
Cu-ATSM) has increased significantly the survival time
of hamsters bearing human GW39 colon cancer tumors. Radiotherapy
experiments were performed in animals bearing either 7-day-old
(0.5–1.0 g) or 15-day-old (1.5–2.0 g) tumors. Studies compared
animals treated with a single dose of 0, 4, 6, 7, 8, or 10 mCi of
64
Cu-ATSM (1 Ci = 37 GBq) with or without the
vasodilator hydralazine. A multiple dose regimen of 3 × 4 mCi at
72-h intervals was studied also. Single doses of >6 mCi of
64
Cu-ATSM and the dose-fractionation protocol significantly
increased the survival time of the hamsters compared with controls. The
highest dose, 10 mCi of
64
Cu-ATSM, increased survival to
135 days in 50% of animals bearing 7-day-old tumors, 6-fold longer
than control animals' survival (20 days), with only transient
leucopenia and thrombocytopenia but no overt toxicity. Human absorbed
doses were calculated from hamster biodistribution; the dose-critical
organs were the lower large intestine (1.43 ± 0.19 rad/mCi) and
upper large intestine (1.20 ± 0.38 rad/mCi). High-resolution
MRI and positron-emission tomography using a therapeutic administration
of 10 mCi were used to monitor tumor volume and morphology and to
assess tumor dosimetry accurately, giving a tumor dose of 81 ±
7.5 rad/mCi.
64
Cu-ATSM has increased the survival time of
tumor-bearing animals significantly with no acute toxicity and thus is
a promising agent for radiotherapy.
Alcohol and upper gastrointestinal tract cancer: the role of local acetaldehyde production
Authors: Homann Nils
Published: July 2002

Alcohol is, together with tobacco smoke, the main cause for upper GI tract cancer in industrialized countries. However, the tumour‐promoting effects of alcohol intake are poorly understood and alcohol itself is not carcinogenic in the animal model. There is increasing evidence that alcohol metabolism, rather than the alcohol itself, generates carcinogenic and cell‐toxic compounds. Acetaldehyde, first metabolite of ethanol, is highly toxic, mutagenic and carcinogenic. Polymorphisms in the genes coding for enzymes responsible for acetaldehyde accumulation and detoxification have been associated with an increased cancer risk. Acetaldehyde can also be produced in the mucosa and by the physiological microflora. This review summarizes the scientific evidence that alcohol intake leads to a local production of acetaldehyde. It describes the role of the oral microflora, the mucosa and the salivary glands in this process and shows that local acetaldehyde production from ethanol may contribute to the carcinogenesis of alcohol intake in the upper GI tract.
Activation of G12/G13 Results in Shape Change and Rho/Rho-Kinase–mediated Myosin Light Chain Phosphorylation in Mouse Platelets
Authors: Klages Birgit , Brandt Ursula , Simon Melvin I. , Schultz Günter , Offermanns Stefan
Published: July 2002

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the α-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Gαq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72syk and stimulation of pp60c-src as well as in phosphorylation of myosin light chain (MLC) in Gαq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Gαq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase–mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase–dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.
The Nonreceptor Tyrosine Kinase Fer Mediates Cross-Talk between N-Cadherin and β1-Integrins
Authors: Arregui Carlos , Pathre Purnima , Lilien Jack , Balsamo Janne
Published: July 2002

Cadherins and integrins must function in a coordinated manner to effectively mediate the cellular interactions essential for development. We hypothesized that exchange of proteins associated with their cytoplasmic domains may play a role in coordinating function. To test this idea, we used Trojan peptides to introduce into cells and tissues peptide sequences designed to compete for the interaction of specific effectors with the cytoplasmic domain of N-cadherin, and assayed their effect on cadherin- and integrin-mediated adhesion and neurite outgrowth. We show that a peptide mimicking the juxtamembrane (JMP) region of the cytoplasmic domain of N-cadherin results in inhibition of N-cadherin and β1-integrin function. The effect of JMP on β1-integrin function depends on the expression of N-cadherin and is independent of transcription or translation. Treatment of cells with JMP results in the release of the nonreceptor tyrosine kinase Fer from the cadherin complex and its accumulation in the integrin complex. A peptide that mimics the first coiled-coil domain of Fer prevents Fer accumulation in the integrin complex and reverses the inhibitory effect of JMP. These findings suggest a new mechanism through which N-cadherin and β1-integrins are coordinately regulated: loss of an effector from the cytoplasmic domain of N-cadherin and gain of that effector by the β1-integrin complex.
Abortive Proliferation of Rare T Cells Induced by Direct or Indirect Antigen Presentation by Rare B Cells In Vivo
Authors: Townsend Sarah E. , Goodnow Christopher C.
Published: July 2002

Antigen-specific B cells are implicated as antigen-presenting cells in memory and tolerance responses because they capture antigens efficiently and localize to T cell zones after antigen capture. It has not been possible, however, to visualize the effect of specific B cells on specific CD4+ helper T cells under physiological conditions. We demonstrate here that rare T cells are activated in vivo by minute quantities of antigen captured by antigen-specific B cells. Antigen-activated B cells are helped under these conditions, whereas antigen-tolerant B cells are killed. The T cells proliferate and then disappear regardless of whether the B cells are activated or tolerant. We show genetically that T cell activation, proliferation, and disappearance can be mediated either by transfer of antigen from antigen-specific B cells to endogenous antigen-presenting cells or by direct B–T cell interactions. These results identify a novel antigen presentation route, and demonstrate that B cell presentation of antigen has profound effects on T cell fate that could not be predicted from in vitro studies.
Infection of Mice with Mycobacterium bovis–Bacillus Calmette-Guérin (BCG) Suppresses Allergen-induced Airway Eosinophilia
Authors: Erb Klaus Josef , Holloway John W. , Sobeck Alexandra , Moll Heidrun , Le Gros Graham
Published: July 2002

It has been proposed that the increase in prevalence and severity of atopic disorders inversely correlates with exposure to infectious diseases such as tuberculosis. We have investigated this issue by combining an intranasal Mycobacterium bovis–Bacillus Calmette-Guérin (BCG) infection with a murine model of allergen, (ovalbumin [OVA]) induced airway eosinophilia. BCG infection either 4 or 12 wk before allergen airway challenge resulted in a 90–95 and 60–70% reduction in eosinophilia within the lungs, respectively, compared to uninfected controls. The inhibition of airway eosinophilia correlated with a reduced level of IL-5 production by T cells from the lymph node draining the site of OVA challenge. Interestingly, BCG infection of the lung had no effect on IgG1 and IgE OVA-specific serum immunoglobulin or blood eosinophil levels. Furthermore, BCG-induced inhibition of airway eosinophilia was strongly reduced in interferon (IFN)-γ receptor–deficient mice and could be partially reversed by intranasal IL-5 application. Intranasal BCG infections could also reduce the degree of lung eosinophilia and IL-5 produced by T cells after Nippostrongylus brasiliensis infection. Taken together, our data suggest that IFN-γ produced during the T helper cell (Th)1 immune response against BCG suppresses the development of local inflammatory Th2 responses in the lung. Most importantly, this inhibition did not extend to the systemic immunoglobulin response against OVA. Our data support the view that mycobacterial infections have the potential to suppress the development of atopic disorders in humans.
CD4+ T Cell–mediated Granulomatous Pathology in Schistosomiasis Is Downregulated by a B Cell–dependent Mechanism Requiring Fc Receptor Signaling
Authors: Jankovic Dragana , Cheever Allen W. , Kullberg Marika C. , Wynn Thomas A. , Yap George , Caspar Patricia , Lewis Fred A. , Clynes Raphael , Ravetch Jeffrey V. , Sher Alan
Published: July 2002

The effector functions of CD4+ T lymphocytes are generally thought to be controlled by distinct populations of regulatory T cells and their soluble products. The role of B cells in the regulation of CD4-dependent host responses is less well understood. Hepatic egg granuloma formation and fibrosis in murine schistosomiasis are dependent on CD4+ lymphocytes, and previous studies have implicated CD8+ T cells or cross-regulatory cytokines produced by T helper (Th) lymphocytes as controlling elements of this pathologic process. In this report, we demonstrate that B cell–deficient (μMT) mice exposed to Schistosoma mansoni develop augmented tissue pathology and, more importantly, fail to undergo the spontaneous downmodulation in disease normally observed during late stages of infection. Unexpectedly, B cell deficiency did not significantly alter T cell proliferative response or cause a shift in the Th1/Th2 balance. Since schistosome-infected Fc receptor–deficient (FcR γ chain knockout) mice display the same exacerbated egg pathology as that observed in infected μMT mice, the B cell– dependent regulatory mechanism revealed by these experiments appears to require receptor-mediated cell triggering. Together, the data demonstrate that humoral immune response/FcR interactions can play a major role in negatively controlling inflammatory disease induced by CD4+ T cells.
APRIL, a New Ligand of the Tumor Necrosis Factor Family, Stimulates Tumor Cell Growth
Authors: Hahne Michael , Kataoka Takao , Schröter Michael , Hofmann Kay , Irmler Martin , Bodmer Jean-Luc , Schneider Pascal , Bornand Tierry , Holler Nils , French Lars E. , Sordat Bernard , Rimoldi Donata , Tschopp Jürg
Published: July 2002

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.
Attenuation of Apoptosis Underlies B Lymphocyte Stimulator Enhancement of Humoral Immune Response
Authors: Do Richard K.G. , Hatada Eunice , Lee Hayyoung , Tourigny Michelle R. , Hilbert David , Chen-Kiang Selina
Published: July 2002

B lymphocyte stimulator (BLyS) is a newly identified monocyte-specific TNF family cytokine. It has been implicated in the development of autoimmunity, and functions as a potent costimulator with antiimmunoglobulin M in B cell proliferation in vitro. Here we demonstrate that BLyS prominently enhances the humoral responses to both T cell–independent and T cell–dependent antigens, primarily by attenuation of apoptosis as evidenced by the prolonged survival of antigen-activated B cells in vivo and in vitro. BLyS acts on primary splenic B cells autonomously, and directly cooperates with CD40 ligand (CD40L) in B cell activation in vitro by protecting replicating B cells from apoptosis. Moreover, although BLyS alone cannot activate the cell cycle, it is sufficient to prolong the survival of naive resting B cells in vitro. Attenuation of apoptosis by BLyS correlates with changes in the ratios between Bcl-2 family proteins in favor of cell survival, predominantly by reducing the proapoptotic Bak and increasing its prosurvival partners, Bcl-2 and Bcl-xL. In either resting or CD40L-activated B cells, the NF-κB transcription factors RelB and p50 are specifically activated, suggesting that they may mediate BLyS signals for B cell survival. Together, these results provide direct evidence for BLyS enhancement of both T cell–independent and T cell–dependent humoral immune responses, and imply a role for BLyS in the conservation of the B cell repertoire. The ability of BLyS to increase B cell survival indiscriminately, at either a resting or activated state, and to cooperate with CD40L, further suggests that attenuation of apoptosis underlies BLyS enhancement of polyclonal autoimmunity as well as the physiologic humoral immune response.
Defective Gp130-Mediated Signal Transducer and Activator of Transcription (Stat) Signaling Results in Degenerative Joint Disease, Gastrointestinal Ulceration, and Failure of Uterine Implantation
Authors: Ernst Matthias , Inglese Melissa , Waring Paul , Campbell Ian K. , Bao Shisan , Clay Fiona J. , Alexander Warren S. , Wicks Ian P. , Tarlinton David M. , Novak Ulrike , Heath Joan K. , Dunn Ashley R.
Published: July 2002

The receptor subunit gp130 transduces multiple cell type–specific activities of the leukemia inhibitory factor (LIF)/interleukin (IL)-6 family of cytokines through the signal transducer and activator of transcription (STAT) and src homology 2 domain–bearing protein tyrosine phosphatase (SHP)-2/ras/Erk pathways. To define STAT-dependent physiological responses, we generated mice with a COOH-terminal gp130ΔSTAT “knock-in” mutation which deleted all STAT-binding sites. gp130ΔSTAT mice phenocopyed mice deficient for IL-6 (impaired humoral and mucosal immune and hepatic acute phase responses) and LIF (failure of blastocyst implantation). However, unlike mice with null mutations in any of the components in the gp130 signaling pathway, gp130ΔSTAT mice also displayed gastrointestinal ulceration and a severe joint disease with features of chronic synovitis, cartilaginous metaplasia, and degradation of the articular cartilage. Mitogenic hyperresponsiveness of synovial cells to the LIF/IL-6 family of cyto-kines was caused by sustained gp130-mediated SHP-2/ras/Erk activation due to impaired STAT-mediated induction of suppressor of cytokine signaling (SOCS) proteins which normally limits gp130 signaling. Therefore, the joint pathology in gp130ΔSTAT mice is likely to arise from the disturbance of the otherwise balanced activation of the SHP-2/ras/Erk and STAT signaling cascades emanating from gp130.
Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits
Authors: McNicholas Carmel M. , Canessa Cecilia M.
Published: July 2002

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na+ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na+ than Li+ currents (INa+/ILi+ 1.2) whereas αγ channels expressed smaller Na+ than Li+ currents (INa+/ILi+ 0.55); (b) the Michaelis Menten constants (Km) of activation of current by increasing concentrations of external Na+ and Li+ of αβ channels were larger (Km > 180 mM) than those of αγ channels (Km of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na+ and 4.2 pS for Li+) were smaller than those of αγ channels (6.5 pS for Na+ and 10.8 pS for Li+); (d) the half-inhibition constant (Ki) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the Ki of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.
𝐶*-algebras associated with branched coverings
Authors: Deaconu Valentin , Muhly Paul
Published: July 2002

In this note we analyze the

C

∗

C^{\ast }

-algebra associated with a branched covering both as a groupoid

C

∗

C^{\ast }

-algebra and as a Cuntz-Pimsner algebra. We determine conditions when the algebra is simple and purely infinite. We indicate how to compute the K-theory of several examples, including one related to rational maps on the Riemann sphere.
On the finiteness of Bass numbers of local cohomology modules
Authors: Kawasaki Ken-ichiroh
Published: July 2002

In this note we show that, if

I
I

is an ideal of dimension 1 of an analytically irreducible local ring, then the Bass numbers of local cohomology modules with support in

V
(
I
)

V(I)

are finite.
Cohomology of the complement of a free divisor
Authors: Castro-Jiménez Francisco , Narváez-Macarro Luis , Mond David
Published: July 2002

We prove that if

D
D

is a “strongly quasihomogeneous" free divisor in the Stein manifold

X
X

, and

U
U

is its complement, then the de Rham cohomology of

U
U

can be computed as the cohomology of the complex of meromorphic differential forms on

X
X

with logarithmic poles along

D
D

, with exterior derivative. The class of strongly quasihomogeneous free divisors, introduced here, includes free hyperplane arrangements and the discriminants of stable mappings in Mather’s nice dimensions (and in particular the discriminants of Coxeter groups).
De Rham cohomology of logarithmic forms on arrangements of hyperplanes
Authors: Wiens Jonathan , Yuzvinsky Sergey
Published: July 2002

The paper is devoted to computation of the cohomology of the complex of logarithmic differential forms with coefficients in rational functions whose poles are located on the union of several hyperplanes of a linear space over a field of characteristic zero. The main result asserts that for a vast class of hyperplane arrangements, including all free and generic arrangements, the cohomology algebra coincides with the Orlik-Solomon algebra. Over the field of complex numbers, this means that the cohomologies coincide with the cohomologies of the complement of the union of the hyperplanes. We also prove that the cohomologies do not change if poles of arbitrary multiplicity are allowed on some of the hyperplanes. In particular, this gives an analogue of the algebraic de Rham theorem for an arbitrary arrangement over an arbitrary field of zero characteristic.
A posteriori error estimates for nonlinear problems. 𝐿^{𝑟}(0,𝑇;𝐿^{𝜌}(Ω))-error estimates for finite element discretizations of parabolic equations
Authors: Verfürth R.
Published: July 2002

Using the abstract framework of [R. Verfürth: A posteriori error estimates for nonlinear problems. Finite element discretizations of elliptic equations. Math. Comput. 62 (206), 445 – 475 (1994)] we analyze a residual a posteriori error estimator for space-time finite element discretizations of quasilinear parabolic pdes. The estimator gives global upper and local lower bounds on the error of the numerical solution. The finite element discretizations in particular cover the so-called

θ
\theta

-scheme, which includes the implicit and explicit Euler methods and the Crank-Nicholson scheme.
Information theoretical probe selection for hybridisation

experiments
Authors: Herwig Ralf , Schmitt Armin O. , Steinfath Matthias , O’Brien John , Seidel Henrik , Meier-Ewert Sebastian , Lehrach Hans , Radelof Uwe
Published: July 2002

Motivation: The choice of probes is an important feature of hybridisation experiments. In this paper we present an algorithm that optimises probes with respect to a training set of sequences based on Shannon entropy as a quality criterion. The practical motivation for our algorithm is oligonucleotide fingerprinting, a method for the simultaneous identification of sequences (cDNA or genomic DNA) by their hybridisation tags according to a set of short probes such as octamers, although the algorithm is of course not restricted to that application.

Results: We can show that our method is superior to the selection of probes according to their frequencies, which is a widely used strategy, and to randomly chosen probe sets. The quality of probe sets is assessed by a simulation pipeline that entails the set of probes as a simulation parameter. The performance of probe sets trained on sequences from different organisms shows additionally that probes should be chosen with regard to the organism under analysis. Case studies are presented on how constraints (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{G\ +\ C}\) \end{document}-content, complexity of the individual probes) influence the selection process.

Availability: A description of the oligonucleotide fingerprinting pipeline is published on our web-page http://www.molgen.mpg.de/~ag_onf/met.htm. An executable of the algorithm and probe lists designed for human and rodents can be downloaded from the ftp-site ftp://ftp.molgen.mpg.de/pub/mpimg/probe_design/.

Contact: herwig@molgen.mpg.de

To whom correspondence should be addressed.
Control of the eukaryotic cell cycle by MAP kinase signaling pathways
Authors: Wilkinson Marc G. , Millar Jonathan B. A.
Published: July 2002

ABSTRACT In an often rapidly changing environment, cells must adapt by monitoring and reacting quickly to extracellular stimuli detected by membrane‐bound receptors and proteins. Reversible phosphorylation of intracellular regulatory proteins has emerged as a crucial mechanism effecting the transmission and modulation of such signals and is determined by the relative activities of protein kinases and phosphatases within the cell. These are often arranged into complex signaling networks that may function independently or be subject to cross‐regulation. Recently, genetic and biochemical analyses have identified the universally conserved mitogen‐activated protein (MAP) kinase cascade as one of the most ubiquitous signal transduction systems. This pathway is activated after a variety of cellular stimuli and regulates numerous physiological processes, particularly the cell division cycle. Progression through the cell cycle is critically dependent on the presence of environmental growth factors and stress stimuli, and failure to correctly integrate such signals into the cell cycle machinery can lead to the accumulation of genetic damage and genomic instability characteristic of cancer cells. Here we focus on the MAP kinase cascade and discuss the molecular mechanisms by which these extensively studied signaling pathways influence cell growth and proliferation.—Wilkinson, M. G., Millar, J. B. A. Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J. 14, 2147‐2157 (2000)
The Effects of Dopamine D1Receptor Blockade in the Prelimbic–Infralimbic Areas on Behavioral Flexibility
Authors: Ragozzino Michael E.
Published: July 2002

This study examined the effects of a dopamine D1antagonist, SCH23390, infused into the prelimbic–infralimbic areas on the acquisition of a response and visual-cue discrimination task, as well as a shift from a response to a visual-cue discrimination and vice versa. Each test was carried out in a cross-maze. The response discrimination required learning to always turn in the same direction (right or left) for a cereal reinforcement. The visual-cue discrimination required learning to always enter the arm with the visual cue. In experiment 1, rats were tested on the response discrimination task, followed by the visual-cue discrimination task. In experiment 2, the testing order was reversed. Bilateral infusions of SCH23390 (0.1 or 1 μg/0.5 μL) into the prelimbic–infralimbic areas did not impair acquisition of the response or visual-cue discrimination tasks. SCH23390 injections at 1 μg, but not 0.1 μg impaired performance when shifting from a response to a visual-cue discrimination, and vice versa. Analysis of the errors revealed that the deficit was due to perseveration of the previously learned strategy. These results suggest that activation of dopamine D1receptors in the prelimbic–infralimbic areas may be critical for the suppression of a previously relevant strategy and/or generating new strategies.
Dosimetry of pulsed clinical proton beams by a small ionization chamber
Authors: Nohtomi A. , Sakae T. , Tsunashima Y. , Kohno R.
Published: July 2002

Response of a micro volume (0.01 ml) ionization chamber has been studied with pulsed proton beams which are used for clinical purposes and has been compared with those of some JARP ionization chambers (0.6 ml). All chambers used had been calibrated by standard beams at the Electrotechnical Laboratory (ETL) and exposure calibration factors, were obtained on advance. Two methods are used to compensate the general recombination which occurs during pulsed beam irradiations: theoretical correction by a Boag's formulation and a modified two‐voltage technique. An evaluation of absolute absorbed dose‐to‐water is performed on the basis of the protocol provided by ICRU report 59. The results imply that, to a first approximation, both chambers indicate the almost same result within 2% when unknown chamber‐dependent parameters of the micro chamber are tentatively assumed to be identical to those of the JARP chamber for the calibration with beams. The about 1.5% discrepancy observed in the response of both chambers is not discussible due to presumably 1–2% uncertainty of the protocol of ICRU report 59 which does not include any chamber‐dependent corrections for the perturbation effects in proton beams.
An analysis of the assumptions and their significance in the determination of required shielding of CT installations
Authors: Harpen Michael D.
Published: July 2002

In this investigation we propose a simple model for the spatial distribution of scattered photons from a CT unit. The model is used to determine the shielding required for a CT installation. A mathematical based spreadsheet which carries out the calculation is given and is also used to assess the significance of the various assumptions used in CT barrier calculations.
Opposite momenta lead to opposite directions
Authors: Peres Asher
Published: July 2002

When a particle decays into two fragments, the wavefunctions of the latter are spherical shells with expanding radii. In spite of this spherical configuration, the two particles can be detected only in opposite directions.
Lorentz transformation of a system carrying “Hidden Momentum”
Authors: Comay E.
Published: July 2002

The relations between an energy–momentum tensor and its corresponding energy–momentum four-vector are discussed. A particular emphasis is put on conditions guaranteeing that spatial integrals of the energy–momentum densities pertain to a true four-vector. Cases where such integrals are not components of a true four-vector are analyzed and the usefulness of the notion of a false four-vector is pointed out. Results are used for explaining Lorentz transformation properties of “hidden momentum.”