bluuebunny/crossref_metadata_2025 huggingface dataset:
Chloroplast small heat shock proteins: Evidence for atypical evolution of an organelle-localized protein
Authors: Waters Elizabeth R. , Vierling Elizabeth
Published: July 2002

Knowledge of the origin and evolution of gene families is critical to our understanding of the evolution of protein function. To gain a detailed understanding of the evolution of the small heat shock proteins (sHSPs) in plants, we have examined the evolutionary history of the chloroplast (CP)-localized sHSPs. Previously, these nuclear-encoded CP proteins had been identified only from angiosperms. This study reveals the presence of the CP sHSPs in a moss,
Funaria hygrometrica
. Two clones for CP sHSPs were isolated from a
F. hygrometrica
heat shock cDNA library that represent two distinct CP sHSP genes. Our analysis of the CP sHSPs reveals unexpected evolutionary relationships and patterns of sequence conservation. Phylogenetic analysis of the CP sHSPs with other plant CP sHSPs and eukaryotic, archaeal, and bacterial sHSPs shows that the CP sHSPs are not closely related to the cyanobacterial sHSPs. Thus, they most likely evolved via gene duplication from a nuclear-encoded cytosolic sHSP and not via gene transfer from the CP endosymbiont. Previous sequence analysis had shown that all angiosperm CP sHSPs possess a methionine-rich region in the N-terminal domain. The primary sequence of this region is not highly conserved in the
F. hygrometrica
CP sHSPs. This lack of sequence conservation indicates that sometime in land plant evolution, after the divergence of mosses from the common ancestor of angiosperms but before the monocot–dicot divergence, there was a change in the selective constraints acting on the CP sHSPs.
Hormone-induced secretory and nuclear translocation of calmodulin: Oscillations of calmodulin concentration with the nucleus as an integrator
Authors: Craske Madeleine , Takeo Teruko , Gerasimenko Oleg , Vaillant Camille , Török Katalin , Petersen Ole H. , Tepikin Alexei V.
Published: July 2002

Many important enzyme activities are regulated by Ca
2+
-dependent interactions with calmodulin (CaM). Some of the most important targets for CaM action are in the nucleus, and Ca
2+
-dependent CaM translocation into this organelle has been reported. Hormone-evoked cytosolic Ca
2+
signals occur physiologically as oscillations, but, so far, oscillations in CaM concentration have not been described. We loaded fluorescent-labeled CaM into pancreatic acinar cells and monitored the fluorescence in various regions by confocal microscopy. Sustained high concentrations of the hormone cholecystokinin or the neurotransmitter acetylcholine evoked a transient movement of cytosolic CaM from the basal nonnuclear area into the secretory granule region and, thereafter, a more substantial and prolonged translocation of CaM into the nucleoplasm. About 50% of the CaM that bound Ca
2+
translocated. At a lower hormone concentration, evoking Ca
2+
oscillations, regular spikes of increased CaM concentration were seen in the secretory granule region with mirror image spikes of decreased CaM concentration in the basal nonnuclear region. The nucleus was able to integrate the Ca
2+
spike-evoked pulses of CaM translocation into a sustained elevation of the nucleoplasmic concentration of this protein.
Endothelin 3 induces the reversion of melanocytes to glia through a neural crest-derived glial-melanocytic progenitor
Authors: Dupin Elisabeth , Glavieux Corinne , Vaigot Pierre , Le Douarin Nicole M.
Published: July 2002

Functional signaling of endothelin 3 (ET3) and its receptor B (ETRB) has been shown to be required for the development of neural crest (NC)-derived pigment cells in mouse, but the precise role of ET3 is not completely understood. Using the avian embryo as a model, we previously reported that ET3 promotes the survival and proliferation of unipotent melanocyte and bipotent glia-melanocyte precursors in trunk NC cultures. Here we investigated whether, at later stages, embryonic pigment cells respond to ET3. Such a possibility is supported by the previous finding that,
in vivo
, avian melanocytes express endothelin receptor B2 (ETRB2) during migration and after their differentiation in the skin. We found that
in vitro
ET3 exerts a dose-dependent stimulation of proliferation and melanogenesis in NC cells that had homed to the epidermis of embryonic quail dorsal skin. Moreover, in clonal cultures of skin-derived pigment cells, ET3 induces rapid cell divisions of clonogenic melanocytes that generate a mixed progeny of melanocytes and cells devoid of pigment granules and expressing glial markers in more than 40% of the colonies. It can therefore be concluded that ET3 is strongly mitogenic to embryonic pigment cells and able to alter their differentiation program, leading them to recapitulate the glial-melanocyte bipotentiality of their NC ancestors.
MouseSeboxhomeobox gene expression in skin, brain, oocytes, and two-cell embryos
Authors: Cinquanta Mario , Rovescalli Alessandra C. , Kozak Christine A. , Nirenberg Marshall
Published: July 2002

Seboxis a mouse paired-like homeobox gene, previously namedOG-9. Seboxgenomic DNA and cDNA were cloned and sequenced. In addition, rat and humanSeboxgenomic DNAs were cloned and sequenced, and the predicted amino acid sequences were compared. The mouseSeboxgene was mapped to chromosome 11 near theEvi 2locus. The mouseSeboxgene is expressed in brain, skin, ovary, and liver of mice. In the brain, theSeboxgene is expressed in the cerebral cortex and CA areas of the hippocampus, pontine nuclei, choroid plexus, and the cerebellum. Northern analysis and RNase protection assays revealed low levels ofSeboxRNA in 12-day mouse embryos and higher levels in 18- and 19-day embryos. In late embryos and newborn mice,Seboxexpression is localized in the epidermis. In adult mice,SeboxRNA was found in maturing oocytes and in fertilized eggs; however, the abundance ofSeboxRNA is decreased in the two-cell embryo, and little or none was detected in the four-cell embryo. Hence,Seboxis a maternally expressed homeobox gene.
A model for individual and collective cell movement in
Dictyostelium
discoideum
Authors: Palsson Eirikur , Othmer Hans G.
Published: July 2002

The cellular slime mold
Dictyostelium discoideum
is a widely used model system for studying a variety of basic processes in development, including cell–cell signaling, signal transduction, pattern formation, cell motility, and the movement of tissue-like aggregates of cells. Many aspects of cell motion are poorly understood, including how individual cell behavior produces the collective motion of cells observed within the mound and slug. Herein, we describe a biologically realistic model for motile
D. discoideum
cells that can generate active forces, that interact via surface molecules, and that can detect and respond to chemotactic signals. We model the cells as deformable viscoelastic ellipsoids and incorporate signal transduction and cell–cell signaling by using a previously developed model. The shape constraint restricts the admissible deformations but makes the simulation of a large number of interacting cells feasible. Because the model is based on known processes, the parameters can be estimated or measured experimentally. We show that this model can reproduce the observations on the chemotactic behavior of single cells, streaming during aggregation, and the collective motion of an aggregate of cells driven by a small group of pacemakers. The model predicts that the motion of two-dimensional slugs [Bonner, J. T. (1998)
Proc. Natl. Acad. Sci. USA
95, 9355–9359] results from the same behaviors that are exhibited by individual cells; it is not necessary to invoke different mechanisms or behaviors. Our computational experiments also suggest previously uncharacterized phenomena that may be experimentally observable.
Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange
Authors: Hoofnagle Andrew N. , Resing Katheryn A. , Goldsmith Elizabeth J. , Ahn Natalie G.
Published: July 2002

Changes in protein mobility accompany changes in conformation
during the trans-activation of enzymes; however, few studies exist that
validate or characterize this behavior. In this study, amide
hydrogen/deuterium exchange/mass spectrometry was used to probe the
conformational flexibility of extracellular signal-regulated protein
kinase-2 before and after activation by phosphorylation. The exchange
data indicated that extracellular regulated protein kinase-2 activation
caused altered backbone flexibility in addition to the conformational
changes previously established by x-ray crystallography. The changes in
flexibility occurred in regions involved in substrate binding and
turnover, suggesting their importance in enzyme regulation.
Defective repression of c-
myc
in breast cancer cells: A loss at the core of the transforming growth factor β growth arrest program
Authors: Chen Chang-Rung , Kang Yibin , Massagué Joan
Published: July 2002

Loss of growth inhibitory responses to the cytokine transforming
growth factor β (TGF-β) in cancer cells may result from mutational
inactivation of TGF-β receptors or their signal transducers, the Smad
transcription factors. In breast cancer, however, loss of TGF-β
growth inhibition often occurs without a loss of these signaling
components. A genome-wide analysis of rapid TGF-β gene responses in
MCF-10A human mammary epithelial cells and MDA-MB-231 breast cancer
cells shows that c-
myc
repression, a response that is
key to the TGF-β program of cell cycle arrest, is selectively lost in
the cancer cell line. Transformation of MCF-10A cells with
c-Ha-
ras
and c
-erbB2
oncogenes also led
to a selective loss of c-
myc
repression and cell cycle
arrest response. TGF-β stimulation of epithelial cells rapidly
induces the formation of a Smad complex that specifically recognizes a
TGF-β inhibitory element in the c-
myc
promoter.
Formation of this complex is deficient in the oncogenically transformed
breast cells. These results suggest that a Smad complex that
specifically mediates c-
myc
repression is a target of
oncogenic signals in breast cancer.
Pupil Mobility, Attainment and Progress During Key Stage 1: A study in cautious interpretation
Authors: Strand Steve
Published: July 2002

Abstract This article explores the association between pupil mobility and attainment in national end of Key Stage 1 (KS1) tests for over 6000 pupils in an English urban education authority. The results indicate that pupil mobility during the early years is associated with significantly lower levels of pupil attainment in reading, writing and mathematics tests at age 7. However, mobile pupils are more likely than stable pupils to be entitled to free school meals, to have English as an additional language, to require higher levels of support in learning English, to have identified and more severe special educational needs and to have higher levels of absence. When the relative impact of these factors is considered, the effect of mobility, while still statistically significant, is substantially reduced. When pupil's baseline assessment results are also included in order to assess educational progress between the age of 4 and 7 years, mobility has a significant effect only on progress in mathematics, and even here the impact is low relative to other pupil background factors. The article concludes that the direct effect of mobility on pupil attainment is likely to be small. However, the implications of mobility for school and classroom management, planning and resourcing are substantial. Examples of good practice and implications for policy are discussed.
Anti‐inflammatory cytokines in asthma and allergy: interleukin‐10, interleukin‐12, interferon‐γ
Authors: Chung Fan
Published: July 2002

Interleukin‐10 (IL‐10) is a cytokine derived from T‐helper type 2 (TH2) cells identified as a suppressor of cytokines from T‐helper type
1(TH1) cells. Interleukin‐12 (IL‐12) is produced by B cells, macrophages and dendritic cells, and primarily regulates TH1 cell differentiation, while suppressing the expansion of TH2 cell clones. Interferon‐γ (IFN‐γ) is a product of TH1 cells and exerts inhibitory effects on TH2 cell differentiation. These cytokines have been implicated in the pathogenesis of asthma and allergies. In this context, IL‐12 and IFN‐γ production in asthma have been found to be decreased, and this may reduce their capacity to inhibit IgE synthesis and allergic inflammation. IL‐10 is a potent inhibitor of monocyte/macrophage function, suppressing the production of many pro‐inflammatory cytokines. A relative underproduction of IL‐10 from alveolar macrophages of atopic asthmatics has been reported. Therapeutic modulation of TH1/TH2 imbalance in asthma and allergy by mycobacterial vaccine, specific immunotherapy and cytoline‐guanosine dinucleotide motif may lead to increases in IL‐12 and IFN‐γ production. Stimulation of IL‐10 production by antigen‐specific T‐cells during immunotherapy may lead to anergy through inhibition of CD28‐costimulatory molecule signalling by IL‐10s anti‐inflammatory effect on basophils, mast cells
and eosinophils.
Interactions of Cocaine with Nimodipine: A Brief Report
Authors: Kosten Thomas R. , Woods Scott W. , Rosen Marc I. , Pearsall H. Rowland
Published: July 2002

This double blind, placebo controlled study of acute calcium channel antagonist use during cocaine administration in five patients found that 60 mg of nimodipine treatment attenuated the systolic, but not diastolic, blood pressure effects of cocaine. In three subjects, a 90 mg dose of nimodipine showed a greater attenuation than that of 60 mg. Subjective effects of cocaine were not altered by either dose of nimodipine.
Trichloroethylene, Trichloroacetic Acid, and Dichloroacetic Acid: Do They Affect Fetal Rat Heart Development?
Authors: Fisher Jeffrey W. , Channel Stephen R. , Eggers Jeffrey S. , Johnson Paula D. , MacMahon Kathleen L. , Goodyear Chuck D. , Sudberry Gregory L. , Warren D. Alan , Latendresse John R. , Graeter Linda J.
Published: July 2002

Trichloroethylene (TCE), trichloroacetic acid (TCA), and dichloroacetic acid (DCA) are commonly found as groundwater contaminants in many regions of the United States. Cardiac birth defects in children have been associated with TCE, and laboratory studies with rodents report an increased incidence of fetal cardiac malformations resulting from maternal exposures to TCE, TCA, and DCA. The objective of this study was to orally treat pregnant CDR(CD) Sprague-Dawley rats with large bolus doses of either TCE (500 mg/kg), TCA (300 mg/kg), or DCA (300 mg/kg) once per day on days 6 through 15 of gestation to determine the effectiveness of these materials to induce cardiac defects in the fetus. All- trans retinoic acid (RA) dissolved in soybean oil was used as a positive control. Soybean oil is commonly used as a dosing vehicle for RA teratology studies and was also used in this study as a dosing vehicle for TCE. Water was used as the dosing vehicle for TCA and DCA. Fetal hearts were examined on gestation day (GD) 21 by an initial in situ, cardiovascular stereomicroscope examination, and then followed by a microscopic dissection and examination of the formalin-fixed heart. The doses selected for TCA and DCA resulted in a modest decrease in maternal weight gain during gestation (3% to 8%). The fetal weights on GD 21 in the TCA and DCA treatment groups were decreased 8% and 9%, respectively, compared to the water control group and 21% in the RA treatment group compared to soybean oil control group. The heart malformation incidence for fetuses from the TCE-, TCA-, and DCA-treated dams did not differ from control values on a per fetus or per litter basis. The rate of heart malformations, on a per fetus basis, ranged from 3% to 5% for TCE, TCA, and DCA treatment groups compared to 6.5% and 2.9% for soybean oil and water control groups. The RA treatment group was significantly higher with 33% of the fetuses displaying heart defects. For TCE, TCA, and DCA treatment groups 42% to 60% of the litters contained at least one fetus with a heart malformation, compared to 52% and 37% of the Utters in the soybean oil and water control groups. For the RA treatment group, 11 of 12 litters contained at least one fetus with a heart malformation. Further research is needed to quantify the spontaneous rates of heart defects for vehicle control rats and to explain the disparity between findings in the present study and other reported findings on the fetal cardiac teratogenicity of TCE, TCA, and DCA.
A Subchronic Toxicity Study of Phosflex 51B in Sprague-Dawley Rats
Authors: Freudenthal Ralph I. , Brandwene David , Clous Welmoed
Published: July 2002

Phosflex 51B is a flame retardant plasticizer that is blended with polyvinyl chloride films to effectively control product flammability. Its composition places it in the butylated triphenyl phosphate category. Previous studies have shown Phosflex 51B to have low acute toxicity, to lack teratogenic and mutagenic activity, and to not induce delayed peripheral neuropathy. The present study was conducted to determine the toxicity of Phosflex 51B after repeated dietary exposure. Four groups, each consisting of 20 male and 20 female Sprague-Dawley rats, received rodent diet containing either 0, 100, 400, or 1600 ppm for 90 days. Parameters measured include body weight, food consumption, clinical observations, hematology, clinical chemistry, and cholinesterase activity. Tissues were examined at necropsy for gross changes and were processed for microscopic pathology. There were no significant treatment-related effects on body weights, food consumption, hematology and clinical chemistry, or cholinesterase values. A significant increase was observed in the absolute and relative mean weights of livers in high-dose male rats, the mean relative fiver weights of the high-dose female animals, the mean relative kidney weights of the high-dose male rats, and the mean absolute weights of the adrenal glands from high-dose female rats. Neither gross nor microscopic pathology examinations revealed tissue changes in these organs or in any other organs. Although increases in fiver, kidney, and adrenal weights were observed in certain animals in the 1600-ppm high-dose group, the administration of Phosflex 51B did not result in significant treatment-related adverse effects at dietary dose levels of 100 and 400 ppm. The no-observable-effect level (NOEL) in this study is 400 ppm.
Effects of Manganese on Inositol Polyphosphate Receptors and Nitric Oxide Synthase Activity in Rat Brain
Authors: Chetty C. S. , Reddy G. R. , Suresh A. , Desaiah D. , Ali S. F. , Slikker W. J.
Published: July 2002

The neurotoxic effects of excessive exposure to manganese (Mn) include degeneration of dopaminergic neurons, impairment of energy metabolism, and perturbations in phosphoinositide (PI) hydrolysis leading to altered calcium (Ca2+) homeostasis. This study is designed to assess the in vitro and in vivo effects of Mn on Ca2+/cahnodulin-dependent neuronal nitric oxide synthase (nNOS) activity and on the regulation of inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) receptors involved in intracellular and extracellular mobilization of Ca2+. In vivo Mn exposure significantly increased 3H-InsP3 and 3H-InsP4 binding in the cerebellum and the cerebral cortex in a dose-dependent manner. However, in vitro Mn decreased 3H-InsP3 binding and increased 3H-InsP4 binding. In vitro and in vivo exposure of Mn inhibited nNOS activity in the cerebellum and the cerebral cortex. Immunohistochemical studies also showed a notable decrease in nNOS immunoreactivity in the granule cell layer of the cerebellum, whereas no significant changes were observed in the cerebral cortex. These data suggest that Mn neurotoxicity may be due to altered calcium homeostasis by its modulation of inositol polyphosphate receptors. Further, the inhibition of nNOS by Mn is of considerable importance because NO regulates a number of neurotransmitter functions.
The Role of Interleukin-6 (IL-6) in Human Sulfur Mustard (HD) Toxicology
Authors: Arroyo Carmen M. , Broomfield Clarence A. , Hackley Brennie E.
Published: July 2002

The authors applied in vitro models of controlled damage to human epidermal keratinocytes (HEKs), human skin fibroblasts (HSFs), and human breast skin tissue (HBST) to examine the mechanism responsible for sulfur mustard (HD)-induced interleukin-6 (IL-6) alterations. Treatment with 100 μM HD for 24 hours resulted in a significant increased amount of IL-6 being secreted by HEKs (HD-exposed to control ratio [E/C] = 4.15 ± 0.07) and by HSFs (E/C = 7.66 ± 0.04). Furthermore, the HD-induced secretion of IL-6 in HEKs was neutralized with monoclonal human IL-6 antibodies. The secretion of IL-6 in HBST supernatant exposed to HD produced conflicting results. Although an increase of IL-6 was observed in control superfusion media from HBST, IL-6 levels were observed to decrease as the concentration of HD increased. Time course of IL-6 mRNA levels were performed using a competitive polymerase chain reaction (PCR) and human IL-6 mRNA assay detection kit in control and HD (100 μM)-treated HEKs cells. IL-6 mRNA transcripts in HD-exposed HEKs were first observed within 2 hours, dropped at 5 to 6 hours, and increased by ® 2.2-fold and 8.5-fold at 24 to 48 hours after HD exposure, respectively, as detected by the Xplore mRNA Quantification System. Surface-enhanced laser desorption ionization (SELDI) mass spectrometry was also applied to study the secretion pattern of IL-6 on lysate preparations of HBST. A peak in the area of 23,194 to 23,226 Da was detected using antibody coupled to the chip. This peak was assigned to correspond to the mass of the IL-6 glycoprotein. Recombinant human IL-6 (rhIL-6) exposed to HD lacked the second disulfide bridge and was partially unfolded, as determined by nuclear magnetic resonance-nuclear Overhauser enhancement and exchange spectroscopy (NMR-NOESY). The disappearance of the resonance peak at 3.54 ppm and the appearance of a new chemical shift at 1.85 ppm suggested that a change in structure had occurred in the presence of HD. From the data, the possibility cannot be excluded that IL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein that indirectly initiates the cascade of events such as skin irritation and blister formation observed in the pathophysiology of HD injury.
Toxicological Studies of a Representative Xerox Reprographic Toner
Authors: Lin George H. Y. , Wilson Joseph C.
Published: July 2002

Typical Xerox reprographic toners consist of a thermoplastic polymer as the major component, a colorant (carbon black or color pigment), and low quantities of additives such as charge control and/or lubricating agents. Another type of Xerox toner contains iron oxides and a polymer as the major components. Among all toners marketed by Xerox Corporation, the original 1075 toner (being discontinued and reformulated) was a major safety concern, because it contained approximately 2% cetylpyridinium chloride (CPC) as a charge control agent. CPC by itself is very toxic and causes severe irritation to the eye and skin. Although CPC has been used in very low concentrations in consumer products such as mouthwash, it was unknown whether a 50-fold dilution of CPC in the toner formulation would represent any safety issue. Therefore, a series of toxicological testing on the original 1075 toner was conducted. The test results indicate that the original Xerox 1075 toner was practically nontoxic following acute oral, dermal, and inhalation exposures; nonirritating to the eye; nonir-ritating/nonsensitizing to the skin; nonmutagenic in a battery of short-term assays (Ames Salmnonella/microsome assay, mouse lym-phoma assay, in vitro sister chromatid exchange assay in Chinese hamster ovarian cells, and in vitro BALB/3T3 cell transformation assay); and nonteratogenic in rats when inhaling the toner dust up to 1.2 g/m3. In addition, no mutagenic responses were observed from testing the urine or feces (by Ames test) and bone marrow (by examining micronucleus formation) of rats exposed to the toner dust at 1.3 g/m3 at the end of a subchronic inhalation study. Because all Xerox toners are alike, the toxicology of the original Xerox 1075 toner was considered a “worst-case” situation, relative to health and safety. However, it did not appear to represent any health and safety issue. The results of this study, together with the fact that no evidence of carcinogenicity was found in the Xerox chronic inhalation study on toner, indicate that Xerox toners are not safety hazards, with respect to the end points indicated in this report.
Identification of the t Complex–encoded Cytoplasmic Dynein Light Chain Tctex1 in Inner Arm I1 Supports the Involvement of Flagellar Dyneins in Meiotic Drive
Authors: Harrison Alistair , Olds-Clarke Patricia , King Stephen M.
Published: July 2002

The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell–specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme.
Dissection of Complex Molecular Interactions of Neurofascin with Axonin-1, F11, and Tenascin-R, Which Promote Attachment and Neurite Formation of Tectal Cells
Authors: Volkmer Hansjürgen , Zacharias Ute , Nörenberg Ursel , Rathjen Fritz G.
Published: July 2002

Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11.

Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment.

In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin– NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays.

In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.
Dap-Kinase Participates in TNF-α–And FAS-Induced Apoptosis and Its Function Requires the Death Domain
Authors: Cohen Ofer , Inbal Boaz , Kissil Joseph L. , Raveh Tal , Berissi Hanna , Spivak-Kroizaman Taly , Feinstein Elena , Kimchi Adi
Published: July 2002

Death-associated protein (DAP)–kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-α and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti–Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-α, Fas, and FADD/MORT1–induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.
The Functional Diversity of Epidermal Keratins Revealed by the Partial Rescue of the Keratin 14 Null Phenotype by Keratin 16
Authors: Paladini Rudolph D. , Coulombe Pierre A.
Published: July 2002

The type I epidermal keratins K14 and K16 are remarkably similar at the primary sequence level. While a structural function has been clearly defined for K14, we have proposed that a function of K16 may be to play a role in the process of keratinocyte activation that occurs after acute injury to stratified epithelia. To compare directly the functions of the two keratins we have targeted the expression of the human K16 cDNA to the progenitor basal layer of the epidermis of K14 null mice. Mice null for K14 blister extensively and die ∼2 d after birth (Lloyd, C., Q.C. Yu, J. Cheng, K. Turksen, L. Degenstein, E. Hutton, and E. Fuchs. 1995. J. Cell Biol. 129:1329–1344). The skin of mice expressing K16 in the absence of K14 developed normally without evidence of blistering. However, as the mice aged they featured extensive alopecia, chronic epidermal ulcers in areas of frequent physical contact, and alterations in other stratified epithelia. Mice expressing a control K16-C14 cDNA also rescue the blistering phenotype of the K14 null mice with only a small percentage exhibiting minor alopecia. While K16 is capable of rescuing the blistering, phenotypic complementation in the resulting skin is incomplete due to the multiple age dependent anomalies. Despite their high sequence similarity, K16 and K14 are not functionally equivalent in the epidermis and other stratified epithelia and it is primarily the carboxy-terminal ∼105 amino acids of K16 that define these differences.
Op18/Stathmin Mediates Multiple Region-Specific Tubulin and Microtubule-Regulating Activities
Authors: Larsson Niklas , Segerman Bo , Howell Bonnie , Fridell Kajsa , Cassimeris Lynne , Gullberg Martin
Published: July 2002

Oncoprotein18/stathmin (Op18) is a regulator of microtubule (MT) dynamics that binds tubulin heterodimers and destabilizes MTs by promoting catastrophes (i.e., transitions from growing to shrinking MTs). Here, we have performed a deletion analysis to mechanistically dissect Op18 with respect to (a) modulation of tubulin GTP hydrolysis and exchange, (b) tubulin binding in vitro, and (c) tubulin association and MT-regulating activities in intact cells. The data reveal distinct types of region-specific Op18 modulation of tubulin GTP metabolism, namely inhibition of nucleotide exchange and stimulation or inhibition of GTP hydrolysis. These regulatory activities are mediated via two-site cooperative binding to tubulin by multiple nonessential physically separated regions of Op18. In vitro analysis revealed that NH2- and COOH-terminal truncations of Op18 have opposite effects on the rates of tubulin GTP hydrolysis. Transfection of human leukemia cells with these two types of mutants result in similar decrease of MT content, which in both cases appeared independent of a simple tubulin sequestering mechanism. However, the NH2- and COOH-terminal–truncated Op18 mutants regulate MTs by distinct mechanisms as evidenced by morphological analysis of microinjected newt lung cells. Hence, mutant analysis shows that Op18 has the potential to regulate tubulin/MTs by more than one specific mechanism.
Homeobox B3 Promotes Capillary Morphogenesis and Angiogenesis
Authors: Myers Connie , Charboneau Aubri , Boudreau Nancy
Published: July 2002

Endothelial cells (EC) express several members of the Homeobox (Hox) gene family, suggesting a role for these morphoregulatory mediators during angiogenesis. We have previously established that Hox D3 is required for expression of integrin αvβ3 and urokinase plasminogen activator (uPA), which contribute to EC adhesion, invasion, and migration during angiogenesis. We now report that the paralogous gene, Hox B3, influences angiogenic behavior in a manner that is distinct from Hox D3. Antisense against Hox B3 impaired capillary morphogenesis of dermal microvascular EC cultured on basement membrane extracellular matrices. Although levels of Hox D3-dependent genes were maintained in these cells, levels of the ephrin A1 ligand were markedly attenuated. Capillary morphogenesis could be restored, however, by addition of recombinant ephrin A1/Fc fusion proteins. To test the impact of Hox B3 on angiogenesis in vivo, we constitutively expressed Hox B3 in the chick chorioallantoic membrane using avian retroviruses that resulted in an increase in vascular density and angiogenesis. Thus, while Hox D3 promotes the invasive or migratory behavior of EC, Hox B3 is required for the subsequent capillary morphogenesis of these new vascular sprouts and, together, these results support the hypothesis that paralogous Hox genes perform complementary functions within a particular tissue type.
Altered Hapten Ligands Antagonize Trinitrophenyl-specific Cytotoxic T Cells and Block Internalization of Hapten-specific Receptors
Authors: Preckel Tobias , Grimm Rudolf , Martin Stefan , Weltzien Hans Ulrich
Published: July 2002

Low molecular chemicals (haptens) frequently cause T cell–mediated adverse immune reactions. Our previous work provided evidence that hapten-specific T cells, in analogy to those specific for nominal peptide antigens, direct their TCR towards hapten-modified, MHC-associated peptides. We now demonstrate that trinitrophenyl (TNP)-specific, class I MHC–restricted CTL from mice may exhibit exquisite specificity for subtle structural details of these hapten determinants, surpassing even the specificity of immunoglobulins. More importantly, these CTL could be antagonized by ligands altered either in their peptide sequence or in their hapten structure. The system was employed to examine the molecular basis of T cell antagonism. Whereas agonists resulted in a dose-dependent downregulation of TCR in different mouse T cell clones, antagonistic peptides totally failed to do so despite engaging the specific TCR. Moreover, simultaneous presentation of antagonist and agonist on the same antigen presenting cell prevented TCR internalization. No signs of anergy or functional receptor inactivation were observed in CTL treated with antagonist-loaded target cells. Based on a serial triggering model of T cell activation, our data favor a model in which antagonists block T cell functions by competitively engaging the specific TCR in unproductive interactions.
Resistance to Leishmania major is Linked to the H2 Region on Chromosome 17 and to Chromosome 9
Authors: Roberts Lynden J. , Baldwin Tracey M. , Curtis Joan M. , Handman Emanuela , Foote Simon J.
Published: July 2002

In Leishmaniasis, as in many infectious diseases, clinical manifestations are determined by the interaction between the genetics of the host and of the parasite. Here we describe studies mapping two loci controlling resistance to murine cutaneous leishmaniasis. Mice infected with L. major show marked genetic differences in disease manifestations: BALB/c mice are susceptible, exhibiting enlarging lesions that progress to systemic disease and death, whereas C57BL/6 are resistant, developing small, self-healing lesions. F2 animals from a C57BL/6 × BALB/c cross showed a continuous distribution of lesion score. Quantitative trait loci (QTL) have been mapped after a non-parametric QTL analysis on a genome-wide scan on 199 animals. QTLs identified were confirmed in a second cross of 271 animals. Linkage was confirmed to a chromosome 9 locus (D9Mit67–D9Mit71) and to a region including the H2 locus on chromosome 17. These have been named lmr2 and lmr1, respectively.
Regulation of ZAP-70 Intracellular Localization: Visualization with the Green Fluorescent Protein
Authors: Sloan-Lancaster Joanne , Zhang Weiguo , Presley John , Williams Brandi L. , Abraham Robert T. , Lippincott-Schwartz Jennifer , Samelson Lawrence E.
Published: July 2002

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.
T Helper 2 (Th2) T Cells Induce Acute Pancreatitis and Diabetes in Immune-compromised Nonobese Diabetic (NOD) Mice
Authors: Pakala Syamasundar V. , Kurrer Michael O. , Katz Jonathan D.
Published: July 2002

Autoimmune diabetes is caused by the CD4+, T helper 1 (Th1) cell-mediated apoptosis of insulin-producing β cells. We have previously shown that Th2 T cells bearing the same T cell receptor (TCR) as the diabetogenic Th1 T cells invade islets in neonatal nonobese diabetic (NOD) mice but fail to cause disease. Moreover, when mixed in excess and cotransferred with Th1 T cells, Th2 T cells could not protect NOD neonates from Th1-mediated diabetes. We have now found, to our great surprise, the same Th2 T cells that produced a harmless insulitis in neonatal NOD mice produced intense and generalized pancreatitis and insulitis associated with islet cell necrosis, abscess formation, and subsequent diabetes when transferred into immunocompromised NOD.scid mice. These lesions resembled allergic inflamation and contained a large eosinophilic infiltrate. Moreover, the Th2-mediated destruction of islet cells was mediated by local interleukin-10 (IL-10) production but not by IL-4. These findings indicate that under certain conditions Th2 T cells may not produce a benign or protective insulitis but rather acute pathology and disease. Additionally, these results lead us to question the feasibility of Th2-based therapy in type I diabetes, especially in immunosuppressed recipients of islet cell transplants.
A Caspase-activated Factor (CAF) Induces Mitochondrial Membrane Depolarization and Cytochrome c Release by a Nonproteolytic Mechanism
Authors: Steemans Margino , Goossens Vera , Van de Craen Marc , Van Herreweghe Franky , Vancompernolle Katia , De Vos Kurt , Vandenabeele Peter , Grooten Johan
Published: July 2002

It is well established that apoptosis is accompanied by activation of procaspases and by mitochondrial changes, such as decrease in mitochondrial transmembrane potential (ΔΨm) and release of cytochrome c. We analyzed the causal relationship between activated caspases and these mitochondrial phenomena. Purified recombinant caspase-1, -11, -3, -6, -7, and -8 were incubated with mitochondria in the presence or absence of additional cellular components, after which ΔΨm was determined. At lower caspase concentrations, only caspase-8 was able to activate a cytosolic factor, termed caspase-activated factor (CAF), which resulted in decrease in ΔΨm and release of cytochrome c. Both CAF-mediated activities could not be blocked by protease inhibitors, including oligopeptide caspase inhibitors. CAF-induced cytochrome c release, but not decrease of ΔΨm, was blocked in mitochondria from cells overexpressing Bcl-2. CAF is apparently involved in decrease of ΔΨm and release of cytochrome c, whereas Bcl-2 only prevents the latter. Hence, CAF may form the link between death domain receptor–dependent activation of procaspase-8 and the mitochondrial events studied.
Angiostatin-mediated Suppression of Cancer Metastases by Primary Neoplasms Engineered to Produce Granulocyte/Macrophage Colony–stimulating Factor
Authors: Dong Zhongyun , Yoneda Junya , Kumar Rakesh , Fidler Isaiah J.
Published: July 2002

We determined whether tumor cells consistently generating granulocyte/macrophage colony– stimulating factor (GM-CSF) can recruit and activate macrophages to generate angiostatin and, hence, inhibit the growth of distant metastasis. Two murine melanoma lines, B16-F10 (syngeneic to C57BL/6 mice) and K-1735 (syngeneic to C3H/HeN mice), were engineered to produce GM-CSF. High GM-CSF (>1 ng/106 cells)– and low GM-CSF (<10 pg/106 cells)–producing clones were identified. Parental, low, and high GM-CSF–producing cells were injected subcutaneously into syngeneic and into nude mice. Parental and low-producing cells produced rapidly growing tumors, whereas the high-producing cells produced slow-growing tumors. Macrophage density inversely correlated with tumorigenicity and directly correlated with steady state levels of macrophage metalloelastase (MME) mRNA. B16 and K-1735 subcutaneous (s.c.) tumors producing high levels of GM-CSF significantly suppressed lung metastasis of 3LL, UV-2237 fibrosarcoma, K-1735 M2, and B16-F10 cells, but parental or low-producing tumors did not. The level of angiostatin in the serum directly correlated with the production of GM-CSF by the s.c. tumors. Macrophages incubated with medium conditioned by GM-CSF– producing B16 or K-1735 cells had higher MME activity and generated fourfold more angiostatin than control counterparts. These data provide direct evidence that GM-CSF released from a primary tumor can upregulate angiostatin production and suppress growth of metastases.
Dendritic Cells Infiltrating Tumors Cotransduced with Granulocyte/Macrophage Colony-Stimulating Factor (Gm-Csf) and Cd40 Ligand Genes Take up and Present Endogenous Tumor-Associated Antigens, and Prime Naive Mice for a Cytotoxic T Lymphocyte Response
Authors: Chiodoni Claudia , Paglia Paola , Stoppacciaro Antonella , Rodolfo Monica , Parenza Mariella , Colombo Mario P.
Published: July 2002

We transduced BALB/c-derived C-26 colon carcinoma cells with granulocyte/macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (CD40L) genes to favor interaction of these cells with host dendritic cells (DCs) and, therefore, cross-priming. Cotransduced cells showed reduced tumorigenicity, and tumor take was followed by regression in some mice. In vivo tumors were heavily infiltrated with DCs that were isolated, phenotyped, and tested in vitro for stimulation of tumor-specific cytotoxic T lymphocytes (CTLs). BALB/c C-26 carcinoma cells express the endogenous murine leukemia virus (MuLV) env gene as a tumor-associated antigen. This antigen is shared among solid tumors of BALB/c and C57BL/6 mice and contains two epitopes, AH-1 and KSP, recognized in the context of major histocompatibility complex class I molecules H-2Ld and H-2Kb, respectively. DCs isolated from C-26/GM/CD40L tumors grown in (BALB/c × C57BL/6)F1 mice (H-2d×b) stimulated interferon γ production by both anti–AH-1 and KSP CTLs, whereas tumor-infiltrating DCs (TIDCs) of BALB/c mice stimulated only anti–AH-1 CTLs. Furthermore, TIDCs primed naive mice for CTL activity as early as 2 d after injection into the footpad, whereas double-transduced tumor cells required at least 5 d for priming; this difference may reflect direct DC priming versus indirect tumor cell priming. Immunohistochemical staining indicated colocalization of DCs and apoptotic bodies in the tumors. These data indicate that DCs infiltrating tumors that produce GM-CSF and CD40L can capture cellular antigens, likely through uptake of apoptotic bodies, and mature in situ to a stage suitable for antigen presentation. Thus, tumor cell–based vaccines engineered to favor the interaction with host DCs can be considered.
Self-Tolerance to the Murine Homologue of a Tyrosinase-Derived Melanoma Antigen
Authors: Colella Teresa A. , Bullock Timothy N.J. , Russell Liane B. , Mullins David W. , Overwijk Willem W. , Luckey Chance John , Pierce Richard A. , Restifo Nicholas P. , Engelhard Victor H.
Published: July 2002

The human tyrosinase-derived peptide YMDGTMSQV is presented on the surface of human histocompatibility leukocyte antigen (HLA)-A*0201+ melanomas and has been suggested to be a tumor antigen despite the fact that tyrosinase is also expressed in melanocytes. To gain information about immunoreactivity and self-tolerance to this antigen, we established a model using the murine tyrosinase-derived homologue of this peptide FMDGTMSQV, together with transgenic mice expressing the HLA-A*0201 recombinant molecule AAD. The murine peptide was processed and presented by AAD similarly to its human counterpart. After immunization with recombinant vaccinia virus encoding murine tyrosinase, we detected a robust AAD-restricted cytotoxic T lymphocyte (CTL) response to FMDGTMSQV in AAD transgenic mice in which the entire tyrosinase gene had been deleted by a radiation-induced mutation. A residual response was observed in the AAD+tyrosinase+ mice after activation under certain conditions. At least some of these residual CTLs in AAD+tyrosinase+ mice were of high avidity and induced vitiligo upon adoptive transfer into AAD+tyrosinase+ hosts. Collectively, these data suggest that FMDGTMSQV is naturally processed and presented in vivo, and that this presentation leads to substantial but incomplete self-tolerance. The relevance of this model to an understanding of the human immune response to tyrosinase is discussed.
The Tprk Protein of Treponema pallidum Is Periplasmic and Is Not a Target of Opsonic Antibody or Protective Immunity
Authors: Hazlett Karsten R.O. , Sellati Timothy J. , Nguyen Tung T. , Cox David L. , Clawson Michael L. , Caimano Melissa J. , Radolf Justin D.
Published: July 2002

The finding that Treponema pallidum, the syphilis spirochete, contains 12 orthologs of the Treponema denticola outer membrane major sheath protein has engendered speculation that members of this T. pallidum repeat (Tpr) family may be similarly surface exposed. In this regard, the TprK protein was reported to be a target of opsonic antibody and protective immunity and subject to immunologically driven sequence variation. Despite these findings, results from our previous analyses of treponemal outer membranes in concert with computer-based predictions for TprK prompted us to examine the cellular location of this protein. TprK–alkaline phosphatase fusions expressed in Escherichia coli demonstrate that TprK contains a signal peptide. However, opsonophagocytosis assays failed to indicate surface exposure of TprK. Moreover, results from three independent methodologies, i.e., (a) indirect immunofluorescence analysis of agarose-encapsulated organisms, (b) proteinase K treatment of intact spirochetes, and (c) Triton X-114 phase partitioning of T. pallidum conclusively demonstrated that native TprK is entirely periplasmic. Consistent with this location, immunization with the recombinant protein failed to induce either protective immunity or select for TprK variants in the rabbit model of experimental syphilis. These findings challenge the notion that TprK will be a component of an efficacious syphilis vaccine.