bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of tumor necrosis factor-deficient mice
Authors: Marino Michael W. , Dunn Ashley , Grail Dianne , Inglese Melissa , Noguchi Yuji , Richards Elizabeth , Jungbluth Achim , Wada Hisashi , Moore Malcolm , Williamson Barbara , Basu Sunanda , Old Lloyd J.
Published: July 2002

Although tumor necrosis factor (TNF) initially came to prominence because of its anti-tumor activity, most attention is now focused on its proinflammatory actions. TNF appears to play a critical role in both early and late events involved in inflammation, from localizing the noxious agent and amplifying the cellular and mediator responses at the local site and systemically, to editing (e.g., apoptosis) injured cells or effete immune cells and repairing inflammatory damage. We have generated mice deficient in TNF (TNF−/−mice) and have begun to examine the multiple functions attributed to TNF. TNF−/−mice develop normally and have no gross structural or morphological abnormalities. As predicted, they are highly susceptible to challenge with an infectious agent (Candida albicans), are resistant to the lethality of minute doses of lipopolysaccharide (LPS) following D-galactosamine treatment, have a deficiency in granuloma development, and do not form germinal centers after immunization. Phagocytic activity of macrophages appears relatively normal, as do T cell functions, as measured by proliferation, cytokine release, and cytotoxicity. B cell response to thymus-independent antigens is normal, but the Ig response to thymus-dependent antigen is reduced. Surprisingly, cytokine production induced by LPS appears essentially intact, with the exception of reduced colony-stimulating factor activity. Other unexpected findings coming from our initial analysis are as follows. (i) TNF has low toxicity in TNF−/−mice. (ii) TNF−/−mice show an anomalous late response to heat-killedCorynebacterium parvum. In contrast to the prompt response (granuloma formation, hepatosplenomegaly) and subsequent resolution phase inC. parvum-injected TNF+/+mice, similarly treated TNF−/−mice show little or no initial response, but then develop a vigorous, disorganized inflammatory response leading to death. These results suggest that TNF has an essential homeostatic role in limiting the extent and duration of an inflammatory process—i.e., an anti-inflammatory function. (iii) In contrast to the expectation that TNF+/+mice and TNF+/−mice would have identical phenotypes, TNF+/−mice showed increased susceptibility to high-dose LPS lethality, increased susceptibility toCandidachallenge, and delayed resolution of theC. parvum-induced inflammatory process, indicating a strong gene dose requirement for different actions of TNF.
Expression of
Sonic hedgehog
gene in regenerating newt limb blastemas recapitulates that in developing limb buds
Authors: Imokawa Yutaka , Yoshizato Katsutoshi
Published: July 2002

This study aimed at characterizing the
Sonic hedgehog
(
shh
) gene in newt limbs, which encodes a signaling molecule of the zone of polarizing activity (ZPA) responsible for determining the anterior–posterior axis of the embryonic chicken and mouse limbs. The reverse transcription–PCR showed that adult newt regenerating limbs express
shh
genes.
In situ
hybridization experiments demonstrated that
shh
genes were expressed in mesenchymal cells of the posterior region of both embryonic buds and regenerating blastemas of newt limbs, strongly suggesting the presence of ZPA in these tissues. Experiments of the axial reversal graft of blastemas further supported this suggestion. The grafted blastemas regenerated supernumerary limbs, and this has been explained by three models: the polar coordinate model, the boundary model, and the polarizing zone model. In favor of the third model, the
shh
gene was expressed not only in the original region (new anterior region) of the graft, but also ectopically in the other region (new posterior region) of the same graft. This study implies that the regenerating limb blastema produces ZPA as the signaling center of the AP patterning as in the developing limb bud and, therefore, supports the notion that the limb regeneration recapitulates the limb development.
Copolymer 1 induces T cells of the T helper type 2 that crossreact with myelin basic protein and suppress experimental autoimmune encephalomyelitis
Authors: Aharoni Rina , Teitelbaum Dvora , Sela Michael , Arnon Ruth
Published: July 2002

The synthetic amino acid copolymer copolymer 1 (Cop 1) suppresses experimental autoimmune encephalomyelitis (EAE) and is beneficial in multiple sclerosis. To further understand Cop 1 suppressive activity, we studied the cytokine secretion profile of various Cop 1-induced T cell lines and clones. Unlike T cell lines induced by myelin basic protein (MBP), which secreted either T cell helper type 1 (Th1) or both Th1 and Th2 cytokines, the T cell lines/clones induced by Cop 1 showed a progressively polarized development toward the Th2 pathway, until they completely lost the ability to secrete Th1 cytokines. Our findings indicate that the polarization of the Cop 1-induced lines did not result from the immunization vehicle or thein vitrogrowing conditions, but rather from the tendency of Cop 1 to preferentially induce a Th2 response. The response of all of the Cop 1 specific lines/clones, which were originated in the (SJL/J×BALB/c)F1hybrids, was restricted to the BALB/c parental haplotype. Even though the Cop 1-induced T cells had not been exposed to the autoantigen MBP, they crossreacted with MBP by secretion of interleukin (IL)-4, IL-6, and IL-10. Administration of these T cellsin vivoresulted in suppression of EAE induced by whole mouse spinal cord homogenate, in which several autoantigens may be involved. Secretion of anti-inflammatory cytokines by Cop 1-induced suppressor cells, in response to either Cop 1 or MBP, may explain the therapeutic effect of Cop 1 in EAE and in multiple sclerosis.
Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta
Authors: Hoggard Nigel , Hunter Leif , Duncan Jacqueline S. , Williams Lynda M. , Trayhurn Paul , Mercer Julian G.
Published: July 2002

Leptin is a 167-aa protein that is secreted from adipose tissue and is important in the regulation of energy balance. It also functions in hematopoiesis and reproduction. To assess whether leptin is involved in fetal growth and development we have examined the distribution of mRNAs encoding leptin and the leptin receptor (which has at least six splice variants) in the 14.5-day postcoitus mouse fetus and in the placenta using reverse transcription–PCR and
in situ
hybridization. High levels of gene expression for leptin, the leptin receptor, and the long splice variant of the leptin receptor with an intracellular signaling domain were observed in the placenta, fetal cartilage/bone, and hair follicles. Receptor expression also was detected in the lung, as well as the leptomeninges and choroid plexus of the fetal brain. Western blotting and immunocytochemistry, using specific antibodies, demonstrated the presence of leptin and leptin receptor protein in these tissues. These results suggest that leptin may play a role in the growth and development of the fetus, both through placental and fetal expression of the leptin and leptin receptor genes. In the fetus, leptin may be multifunctional and have both paracrine and endocrine effects.
Activating mutations for the Met tyrosine kinase receptor in human cancer
Authors: Jeffers Michael , Schmidt Laura , Nakaigawa Noboru , Webb Craig P. , Weirich Gregor , Kishida Takeshi , Zbar Berton , Vande Woude George F.
Published: July 2002

Recently, mutations in the Met tyrosine kinase receptor have been identified in both hereditary and sporadic forms of papillary renal carcinoma. We have introduced the corresponding mutations into the
met
cDNA and examined the effect of each mutation in biochemical and biological assays. We find that the Met mutants exhibit increased levels of tyrosine phosphorylation and enhanced kinase activity toward an exogenous substrate when compared with wild-type Met. Moreover, NIH 3T3 cells expressing mutant Met molecules form foci
in vitro
and are tumorigenic in nude mice. Enzymatic and biological differences were evident among the various mutants examined, and the somatic mutations were generally more active than those of germ-line origin. A strong correlation between the enzymatic and biological activity of the mutants was observed, indicating that tumorigenesis by Met is quantitatively related to its level of activation. These results demonstrate that the Met mutants originally identified in human papillary renal carcinoma are oncogenic and thus are likely to play a determinant role in this disease, and these results raise the possibility that activating Met mutations also may contribute to other human malignancies.
Expansionin vitroof adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability
Authors: Miller Cindy L. , Eaves Connie J.
Published: July 2002

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold,P< 0.001)in vitroof cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1+lin−adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11, flt3-ligand, and Steel factor. Moreover, the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures, long-term culture-initiating cells increased 7- ± 2-fold, myeloid colony-forming cells increased 140- ± 36-fold, and total nucleated cells increased 230- ± 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1+lin−marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension, suggesting that most had undergone at least one self-renewal divisionin vitro. No expansion of stem cells was seen when Sca-1+TER119−CD34+day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult.
Tobacco smoke is a source of toxic reactive glycation products
Authors: Cerami Carla , Founds Hank , Nicholl Iain , Mitsuhashi Tomoko , Giordano Donna , Vanpatten Sonya , Lee Annette , Al-Abed Yousef , Vlassara Helen , Bucala Richard , Cerami Anthony
Published: July 2002

Smokers have a significantly higher risk for developing coronary and cerebrovascular disease than nonsmokers. Advanced glycation end products (AGEs) are reactive, cross-linking moieties that form from the reaction of reducing sugars and the amino groups of proteins, lipids, and nucleic acids. AGEs circulate in high concentrations in the plasma of patients with diabetes or renal insufficiency and have been linked to the accelerated vasculopathy seen in patients with these diseases. Because the curing of tobacco takes place under conditions that could lead to the formation of glycation products, we examined whether tobacco and tobacco smoke could generate these reactive species that would increase AGE formation
in vivo
. Our findings show that reactive glycation products are present in aqueous extracts of tobacco and in tobacco smoke in a form that can rapidly react with proteins to form AGEs. This reaction can be inhibited by aminoguanidine, a known inhibitor of AGE formation. We have named these glycation products “glycotoxins.” Like other known reducing sugars and reactive glycation products, glycotoxins form smoke, react with protein, exhibit a specific fluorescence when cross-linked to proteins, and are mutagenic. Glycotoxins are transferred to the serum proteins of human smokers. AGE-apolipoprotein B and serum AGE levels in cigarette smokers were significantly higher than those in nonsmokers. These results suggest that increased glycotoxin exposure may contribute to the increased incidence of atherosclerosis and high prevalence of cancer in smokers.
Large-scale temporal gene expression mapping of central nervous system development
Authors: Wen Xiling , Fuhrman Stefanie , Michaels George S. , Carr Daniel B. , Smith Susan , Barker Jeffery L. , Somogyi Roland
Published: July 2002

We used reverse transcription–coupled PCR to produce a high-resolution temporal map of fluctuations in mRNA expression of 112 genes during rat central nervous system development, focusing on the cervical spinal cord. The data provide a temporal gene expression “fingerprint” of spinal cord development based on major families of inter- and intracellular signaling genes. By using distance matrices for the pair-wise comparison of these 112 temporal gene expression patterns as the basis for a cluster analysis, we found five basic “waves” of expression that characterize distinct phases of development. The results suggest functional relationships among the genes fluctuating in parallel. We found that genes belonging to distinct functional classes and gene families clearly map to particular expression profiles. The concepts and data analysis discussed herein may be useful in objectively identifying coherent patterns and sequences of events in the complex genetic signaling network of development. Functional genomics approaches such as this may have applications in the elucidation of complex developmental and degenerative disorders.
Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects
Authors: Baumgart Martin , Moos Verena , Schuhbauer Diana , Müller Brigitte
Published: July 2002

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)–Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2qwas determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Aband I-Ekmolecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+BMDMs for 5–8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aqexpression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by anin vitrorestimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.
Single-nucleotide polymorphism in the human mu opioid receptor gene alters β-endorphin binding and activity: Possible implications for opiate addiction
Authors: Bond Cherie , LaForge K. Steven , Tian Mingting , Melia Dorothy , Zhang Shengwen , Borg Lisa , Gong Jianhua , Schluger James , Strong Judith A. , Leal Suzanne M. , Tischfield Jay A. , Kreek Mary Jeanne , Yu Lei
Published: July 2002

Opioid drugs play important roles in the clinical management of pain, as well as in the development and treatment of drug abuse. The mu opioid receptor is the primary site of action for the most commonly used opioids, including morphine, heroin, fentanyl, and methadone. By sequencing DNA from 113 former heroin addicts in methadone maintenance and 39 individuals with no history of drug or alcohol abuse or dependence, we have identified five different single-nucleotide polymorphisms (SNPs) in the coding region of the mu opioid receptor gene. The most prevalent SNP is a nucleotide substitution at position 118 (A118G), predicting an amino acid change at a putative N-glycosylation site. This SNP displays an allelic frequency of approximately 10% in our study population. Significant differences in allele distribution were observed among ethnic groups studied. The variant receptor resulting from the A118G SNP did not show altered binding affinities for most opioid peptides and alkaloids tested. However, the A118G variant receptor binds β-endorphin, an endogenous opioid that activates the mu opioid receptor, approximately three times more tightly than the most common allelic form of the receptor. Furthermore, β-endorphin is approximately three times more potent at the A118G variant receptor than at the most common allelic form in agonist-induced activation of G protein-coupled potassium channels. These results show that SNPs in the mu opioid receptor gene can alter binding and signal transduction in the resulting receptor and may have implications for normal physiology, therapeutics, and vulnerability to develop or protection from diverse diseases including the addictive diseases.
Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow
Authors: Frenette Paul S. , Subbarao Sangeetha , Mazo Irina B. , von Andrian Ulrich H. , Wagner Denisa D.
Published: July 2002

The adhesive mechanisms allowing hematopoietic progenitor cells (HPC) homing to the bone marrow (BM) after BM transplantation are poorly understood. We investigated the role of endothelial selectins and vascular cell adhesion molecule-1 (VCAM-1) in this process. Lethally irradiated recipient mice deficient in both P-and E-selectins (P/E−/−), reconstituted with minimal numbers (≤5 × 10
4
) of wild-type BM cells, poorly survived the procedure compared with wild-type recipients. Excess mortality in P/E−/− mice, after a lethal dose of irradiation, was likely caused by a defect of HPC homing. Indeed, we observed that the recruitment of HPC to the BM was reduced in P/E−/− animals, either splenectomized or spleen-intact. Homing into the BM of P/E−/− recipient mice was further compromised when a function-blocking VCAM-1 antibody was administered. Circulating HPC, 14 hr after transplantation, were greatly increased in P/E−/− mice treated with anti-VCAM-1 compared with P/E−/− mice treated with just IgG or wild-type mice treated with either anti-VCAM-1 or IgG. Our results indicate that endothelial selectins play an important role in HPC homing to the BM. Optimal recruitment of HPC after lethal doses of irradiation requires the combined action of both selectins and VCAM-1 expressed on endothelium of the BM.
Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta
Authors: Pereira Ruth F. , O’Hara Michael D. , Laptev Alexey V. , Halford Kenneth W. , Pollard Marea D. , Class Reiner , Simon Daniela , Livezey Kristin , Prockop Darwin J.
Published: July 2002

Marrow stromal cells from wild-type mice were infused into transgenic mice that had a phenotype of fragile bones resembling osteogenesis imperfecta because they expressed a human minigene for type I collagen. In mice that were irradiated with potentially lethal levels (700 cGy) or sublethal levels (350 cGy), DNA from the donor marrow stromal cells was detected consistently in marrow, bone, cartilage, and lung either 1 or 2.5 mo after the infusions. The DNA also was detected but less frequently in the spleen, brain, and skin. There was a small but statistically significant increase in both collagen content and mineral content of bone 1 mo after the infusion. Similar results were obtained with infusion of relatively large amounts of wild-type whole marrow cells into the transgenic mice. In experiments in which male marrow stromal cells were infused into a female osteogenesis imperfecta-transgenic mouse, fluorescense
in situ
hybridization assays for the Y chromosome indicated that, after 2.5 mo, donor male cells accounted for 4–19% of the fibroblasts or fibroblast-like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, and skin. In a parallel experiment in which whole marrow cells from a male mouse were infused into a female immunodeficient
rag
-
2
mouse, donor male cells accounted for 4–6% of the fibroblasts or fibroblast-like cells in primary cultures. The results support previous suggestions that marrow stromal cells or related cells in marrow serve as a source for continual renewal of cells in a number of nonhematopoietic tissues.
Differential gene expression between developing queens and workers in the honey bee,Apismellifera
Authors: Evans Jay D. , Wheeler Diana E.
Published: July 2002

Many insects show polyphenisms, or alternative morphologies, which are based on differential gene expression rather than genetic polymorphism. Queens and workers are alternative forms of the adult female honey bee and represent one of the best known examples of insect polyphenism. Hormonal regulation of caste determination in honey bees has been studied in detail, but little is known about the proximate molecular mechanisms underlying this process, or any other such polyphenism. We report the success of a molecular-genetic approach for studying queen- and worker-specific gene expression in the development of the honey bee (Apis mellifera). Numerous genes appear to be differentially expressed between the two castes. Seven differentially expressed loci described here belong to at least five distinctly different evolutionary and functional groups. Two are particularly promising as potential regulators of caste differentiation. One is homologous to a widespread class of proteins that bind lipids and other hydrophobic ligands, including retinoic acid. The second locus shows sequence similarity to a DNA-binding domain in the Ets family of transcription factors. The remaining loci appear to be involved with downstream changes inherent to queen- or worker-specific developmental pathways. Caste determination in honey bees is typically thought of as primarily queen determination; our results make it clear that the process involves specific activation of genes in workers as well as in queens.
DNA methylation is a reversible biological signal
Authors: Ramchandani Shyam , Bhattacharya Sanjoy K. , Cervoni Nadia , Szyf Moshe
Published: July 2002

The pattern of DNA methylation plays an important role in regulating different genome functions. To test the hypothesis that DNA methylation is a reversible biochemical process, we purified a DNA demethylase from human cells that catalyzes the cleavage of a methyl residue from 5-methyl cytosine and its release as methanol. We show that similar to DNA methyltransferase, DNA demethylase shows CpG dinucleotide specificity, can demethylate mdCpdG sites in different sequence contexts, and demethylates both fully methylated and hemimethylated DNA. Thus, contrary to the commonly accepted model, DNA methylation is a reversible signal, similar to other physiological biochemical modifications.
Expression of dominant-negative and mutant isoforms of the antileukemic transcription factor Ikaros in infant acute lymphoblastic leukemia
Authors: Sun Lei , Heerema Nyla , Crotty Lisa , Wu Xian , Navara Christopher , Vassilev Alexei , Sensel Martha , Reaman Gregory H. , Uckun Fatih M.
Published: July 2002

Ikaros, a zinc finger-containing DNA-binding protein, is required for normal lymphocyte development, and germline mutant mice that express only non-DNA binding dominant-negative “leukemogenic” Ikaros isoforms lacking critical N-terminal zinc fingers develop an aggressive form of lymphoblastic leukemia 3–6 months after birth. Therefore, we sought to determine whether molecular abnormalities involving the
Ikaros
gene could contribute to the development of acute lymphoblastic leukemia (ALL) in infants. Primary leukemic cells were freshly obtained from 12 infants (<1 year of age) with newly diagnosed ALL. In leukemic cells from each of the 12 infants with ALL, we found high level expression of dominant-negative isoforms of Ikaros with abnormal subcellular compartmentalization patterns. PCR cloning and nucleotide sequencing were used to identify the specific Ikaros isoforms and detect
Ikaros
gene mutations in these cells. Leukemic cells from seven of seven infants with ALL, including five of five MLL-AF4
+
infants, expressed dominant-negative Ikaros isoforms Ik-4, Ik-7, and Ik-8 that lack critical N-terminal zinc fingers. In six of seven patients, we detected a specific mutation leading to an in-frame deletion of 10 amino acids (Δ KSSMPQKFLG) upstream of the transcription activation domain adjacent to the C-terminal zinc fingers of Ik-2, Ik-4, Ik-7, and Ik-8. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal nuclear localization were found in normal infant bone marrow cells and infant thymocytes. These results implicate the expression of dominant-negative Ikaros isoforms and the disruption of normal
Ikaros
function in the leukemogenesis of ALL in infants.
Mechanism by which the IFN-β enhanceosome activates transcription
Authors: Yie Junming , Senger Kate , Thanos Dimitris
Published: July 2002

We demonstrate that in contrast to previous findings by using simple synthetic promoters or activators, the natural IFN-β enhanceosome activates transcription by causing a dramatic increase of the rate by which preinitiation complexes assemble at the promoter. This effect totally depends on the recruitment of the CBP-PolII holoenzyme by the enhanceosome, because its depletion from the extract decelerates the rate of transcription. However, addition of the CBP-PolII holoenzyme back to these extracts fully restores the speed by which the enhanceosome activates transcription. Strikingly, preincubation of the enhanceosome with the CBP-RNA PolII holoenzyme complex results in instant assembly of preinitiation complexes. In contrast, individual IFN-β gene activators function solely by increasing the number of functional preinitiation complexes and not the rate of their assembly. Thus, fast recruitment of the CBP-RNA PolII holoenzyme complex is critical for the rapid activation of IFN-β gene expression by virus infection.
Cocaine- and amphetamine-regulated transcript in the rat vagus nerve: A putative mediator of cholecystokinin-induced satiety
Authors: Broberger Christian , Holmberg Kristina , Kuhar Michael J. , Hökfelt Tomas
Published: July 2002

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the central nervous system. Recent studies have pointed to a role for CART-derived peptides in inhibiting feeding behavior. Although these actions have generally been attributed to hypothalamic CART, it remains to be determined whether additional CART pathways exist that link signals from the gastrointestinal tract to the central control of food intake. In the present study, we have investigated the presence of CART in the rat vagus nerve and nodose ganglion. In the viscerosensory nodose ganglion, half of the neuron profiles expressed CART and its predicted peptide, as determined by
in situ
hybridization and immunohistochemistry. CART expression was markedly attenuated after vagotomy, but no modulation was observed after food restriction or high-fat regimes. A large proportion of CART-labeled neuron profiles also expressed cholecystokinin A receptor mRNA. CART-peptide-like immunoreactivity was transported in the vagus nerve and found in a dense fiber plexus in the nucleus tractus solitarii. Studies on CART in the spinal somatosensory system revealed strong immunostaining of the dorsal horn but only a small number of stained cell bodies in dorsal root ganglia. The present results suggest that CART-derived peptides are present in vagal afferent neurons sensitive to cholecystokinin, suggesting that the role of these peptides in feeding may be explained partly by mediating postprandial satiety effects of cholecystokinin.
HSP70 homolog functions in cell-to-cell movement of a plant virus
Authors: Peremyslov Valery V. , Hagiwara Yuka , Dolja Valerian V.
Published: July 2002

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.
Target genes of β-catenin–T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas
Authors: Mann B. , Gelos M. , Siedow A. , Hanski M. L. , Gratchev A. , Ilyas M. , Bodmer W. F. , Moyer M. P. , Riecken E. O. , Buhr H. J. , Hanski C.
Published: July 2002

Mutations in the adenomatous polyposis coli or β-catenin
gene lead to cytosolic accumulation of β-catenin and, subsequently,
to increased transcriptional activity of the β-catenin–T
cell-factor/lymphoid-enhancer-factor complex. This process seems to
play an essential role in the development of most colorectal
carcinomas. To identify genes activated by β-catenin overexpression,
we used colorectal cell lines for transfection with the β-catenin
gene and searched for genes differentially expressed in the
transfectants. There are four genes affected by β-catenin
overexpression; three overexpressed genes code for two components of
the AP-1 transcription complex, c
-jun
and
fra-1
, and for the urokinase-type plasminogen activator
receptor (uPAR), whose transcription is activated by AP-1. The direct
interaction of the β-catenin–T
cell-factor/lymphoid-enhancer-factor complex with the promoter region
of c
-jun
and
fra-1
was shown in a gel
shift assay. The concomitant increase in β-catenin expression and the
amount of uPAR was confirmed in primary colon carcinomas and their
liver metastases at both the mRNA and the protein levels. High
expression of β-catenin in transfectants, as well as in additionally
analyzed colorectal cell lines, was associated with decreased
expression of ZO-1, which is involved in epithelial polarization. Thus,
accumulation of β-catenin indirectly affects the expression of uPAR
in vitro
and
in vivo
. Together with the
other alterations, β-catenin accumulation may contribute to the
development and progression of colon carcinoma both by
dedifferentiation and through proteolytic activity.
Preferential Zn
2+
influx through Ca
2+
-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production
Authors: Sensi Stefano L. , Yin Hong Z. , Carriedo Sean G. , Rao Shyam S. , Weiss John H.
Published: July 2002

Synaptically released Zn
2+
can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn
2+
-sensitive probe, Newport green, examined levels of [Zn
2+
]
i
attained in cultured cortical neurons on exposure to
N-
methyl-
d
-asparte, kainate, or high K
+
(to activate voltage-sensitive Ca
2+
channels) in the presence of 300 μM Zn
2+
. Indicating particularly high permeability through Ca
2+
-permeable α-amino3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate (Ca-A/K) channels, micromolar [Zn
2+
]
i
rises were observed only after kainate exposures and only in neurons expressing these channels [Ca-A/K(+) neurons]. Further studies using the oxidation-sensitive dye, hydroethidine, revealed Zn
2+
-dependent reactive oxygen species (ROS) generation that paralleled the [Zn
2+
]
i
rises, with rapid oxidation observed only in the case of Zn
2+
entry through Ca-A/K channels. Indicating a mitochondrial source of this ROS generation, hydroethidine oxidation was inhibited by the mitochondrial electron transport blocker, rotenone. Additional evidence for a direct interaction between Zn
2+
and mitochondria was provided by the observation that the Zn
2+
entry through Ca-A/K channels triggered rapid mitochondrial depolarization, as assessed by using the potential-sensitive dye tetramethylrhodamine ethylester. Whereas Ca
2+
influx through Ca-A/K channels also triggers ROS production, the [Zn
2+
]
i
rises and subsequent ROS production are of more prolonged duration.
Essential role of STAT3 for embryonic stem cell pluripotency
Authors: Raz Regina , Lee Chien-Kuo , Cannizzaro Linda A. , d’Eustachio Peter , Levy David E.
Published: July 2002

Propagation of mouse embryonic stem (ES) cells
in vitro
requires exogenous leukemia inhibitory factor (LIF) or related cytokines. Potential downstream effectors of the LIF signal in ES cells include kinases of the Src, Jak, and mitogen-activated protein families and the signal transducer and transcriptional activator STAT3. Activation of nuclear STAT3 and the ability of ES cells to grow as undifferentiated clones were monitored during LIF withdrawal. A correlation was found between levels of STAT3 activity and maintenance of an undifferentiated phenotype at clonal density. In contrast, variation in STAT3 activity did not affect cell proliferation. The requirement for STAT3 was analyzed by targeted mutagenesis in ES cell lines exhibiting different degrees of LIF dependency. An insertional mutation was devised that abrogated
Stat3
gene expression but could be reversed by Cre recombination-mediated excision. ES cells heterozygous for the
Stat3
mutation could be isolated only from E14 cells, the line least dependent on LIF for self-renewal. Targeted clones isolated from other ES cell lines were invariably trisomic for chromosome 11, which carries the
Stat3
locus, and retained normal levels of activated STAT3. Cre-regulated reduction of
Stat3
gene copy number in targeted, euploid E14 clones resulted in dose-dependent losses of STAT3 activity and the efficiency of self-renewal without commensurate changes in cell cycle progression. These results demonstrate an essential role for a critical amount of STAT3 in the maintenance of an undifferentiated ES cell phenotype.
Epidermal growth factor system regulates mucin production in airways
Authors: Takeyama Kiyoshi , Dabbagh Karim , Lee Heung-Man , Agustí Carlos , Lausier James A. , Ueki Iris F. , Grattan Kathleen M. , Nadel Jay A.
Published: July 2002

Goblet-cell hyperplasia is a critical pathological feature in hypersecretory diseases of airways. However, the underlying mechanisms are unknown, and no effective therapy exists. Here we show that stimulation of epidermal growth factor receptors (EGF-R) by its ligands, EGF and transforming growth factor α (TGFα), causes
MUC5AC
expression in airway epithelial cells both in
in vitro
and
in vivo
. We found that a
MUC5AC
-inducing epithelial cell line, NCI-H292, expresses EGF-R constitutively;
EGF-R
gene expression was stimulated further by tumor necrosis factor α (TNFα). EGF-R ligands increased the expression of
MUC5AC
at both gene and protein levels, and this effect was potentiated by TNFα. Selective EGF-R tyrosine kinase inhibitors blocked
MUC5AC
expression induced by EGF-R ligands. Pathogen-free rats expressed little EGF-R protein in airway epithelial cells; intratracheal instillation of TNFα induced EGF-R in airway epithelial cells, and subsequent instillation of EGF-R ligands increased the number of goblet cells, Alcian blue–periodic acid–Schiff staining (reflecting mucous glycoconjugates), and
MUC5AC
gene expression, whereas TNFα, EGF, or TGFα alone was without effect. In sensitized rats, three intratracheal instillations of ovalbumin resulted in EGF-R expression and goblet-cell production in airway epithelium. Pretreatment with EGF-R tyrosine kinase inhibitor, BIBX1522, prevented goblet-cell production both in rats stimulated by TNFα-EGF-R ligands and in an asthma model. These findings suggest potential roles for inhibitors of the EGF-R cascade in hypersecretory diseases of airways.
Prevention of experimental antiphospholipid syndrome and endothelial cell activation by synthetic peptides
Authors: Blank Miri , Shoenfeld Yehuda , Cabilly Shmuel , Heldman Yehudit , Fridkin Mati , Katchalski-Katzir Ephraim
Published: July 2002

Antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, repeated thromboembolic phenomena, and thrombocytopenia. The syndrome is believed to be caused by antiphospholipid beta-2-glycoprotein-I (β2GPI)-dependent Abs or anti-β2GPI Abs by themselves. Using a hexapeptide phage display library, we identified three hexapeptides that react specifically with the anti-β2GPI mAbs ILA-1, ILA-3, and H-3, which cause endothelial cell activation and induce experimental APS. To enhance the binding of the peptides to the corresponding mAbs, the peptides were lengthened to correspond with the site of the β2GPI epitope being recognized by these mAbs. As a result, the following three peptides were prepared: A, NTLKTPRVGGC, which binds to ILA-1 mAb; B, KDKATFGCHDGC, which binds to ILA-3 mAb; and C, CATLRVYKGG, which binds to H-3 mAb. Peptides A, B, and C specifically inhibit bothin vitroandin vivothe biological functions of the corresponding anti-β2GPI mAbs. Exposure of endothelial cells to anti-β2GPI mAbs and their corresponding peptides led to the inhibition of endothelial cell activation, as shown by decreased expression of adhesion molecules (E-selectin, ICAM-1, VCAM-1) and monocyte adhesion.In vivoinfusion of each of the anti-β2GPI mAbs into BALB/c mice, followed by administration of the corresponding specific peptides, prevented the peptide-treated mice from developing experimental APS. The use of synthetic peptides that focus on neutralization of pathogenic anti-β2GPI Abs represents a possible new therapeutic approach to APS.
Epidermal growth factor-induced nuclear factor κB activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells
Authors: Biswas Debajit K. , Cruz Antonio P. , Gansberger Eva , Pardee Arthur B.
Published: July 2002

The epidermal growth factor (EGF) family of receptors (EGFR) is overproduced in estrogen receptor (ER) negative (−) breast cancer cells. An inverse correlation of the level of EGFR and ER is observed between ER− and ER positive (+) breast cancer cells. A comparative study with EGFR-overproducing ER− and low-level producing ER+ breast cancer cells suggests that EGF is a major growth-stimulating factor for ER− cells. An outline of the pathway for the EGF-induced enhanced proliferation of ER− human breast cancer cells is proposed. The transmission of mitogenic signal induced by EGF–EGFR interaction is mediated via activation of nuclear factor κB (NF-κB). The basal level of active NF-κB in ER− cells is elevated by EGF and inhibited by anti-EGFR antibody (EGFR-Ab), thus qualifying EGF as a NF-κB activation factor. NF-κB transactivates the cell-cycle regulatory protein, cyclin D1, which causes increased phosphorylation of retinoblastoma protein, more strongly in ER− cells. An inhibitor of phosphatidylinositol 3 kinase, Ly294–002, blocked this event, suggesting a role of the former in the activation of NF-κB by EGF. Go6976, a well-characterized NF-κB inhibitor, blocked EGF-induced NF-κB activation and up-regulation of cell-cycle regulatory proteins. This low molecular weight compound also caused apoptotic death, predominantly more in ER− cells. Thus Go6976 and similar NF-κB inhibitors are potentially novel low molecular weight therapeutic agents for treatment of ER− breast cancer patients.
Importance of replication in microarray gene expression studies: Statistical methods and evidence from repetitive cDNA hybridizations
Authors: Lee Mei-Ling Ting , Kuo Frank C. , Whitmore G. A. , Sklar Jeffrey
Published: July 2002

We present statistical methods for analyzing replicated cDNA microarray expression data and report the results of a controlled experiment. The study was conducted to investigate inherent variability in gene expression data and the extent to which replication in an experiment produces more consistent and reliable findings. We introduce a statistical model to describe the probability that mRNA is contained in the target sample tissue, converted to probe, and ultimately detected on the slide. We also introduce a method to analyze the combined data from all replicates. Of the 288 genes considered in this controlled experiment, 32 would be expected to produce strong hybridization signals because of the known presence of repetitive sequences within them. Results based on individual replicates, however, show that there are 55, 36, and 58 highly expressed genes in replicates 1, 2, and 3, respectively. On the other hand, an analysis by using the combined data from all 3 replicates reveals that only 2 of the 288 genes are incorrectly classified as expressed. Our experiment shows that any single microarray output is subject to substantial variability. By pooling data from replicates, we can provide a more reliable analysis of gene expression data. Therefore, we conclude that designing experiments with replications will greatly reduce misclassification rates. We recommend that at least three replicates be used in designing experiments by using cDNA microarrays, particularly when gene expression data from single specimens are being analyzed.
The human
DINB1
gene encodes the DNA polymerase Polθ
Authors: Johnson Robert E. , Prakash Satya , Prakash Louise
Published: July 2002

The human
DINB1
gene shares a high degree of homology with the
Escherichia coli
dinB
gene. Here, we purify the
hDINB1-
encoded protein and show that it is a DNA polymerase. Because hDinB1 is the eighth eukaryotic DNA polymerase to be described, we have named it DNA polymerase (Pol) θ. hPolθ is unable to bypass a
cis-syn
thymine–thymine dimer, nor does it bypass a (6–4) photoproduct or an abasic site. We also examine the fidelity of hPolθ on nondamaged DNA templates by steady-state kinetic analyses and find that hPolθ misincorporates deoxynucleotides with a frequency of about 10
−3
to 10
−4
. We discuss the relationship between the fidelity of hPolθ and its inability to bypass DNA damage.
Functional relationships in the nuclear and extended family: A 16‐culture study
Authors: Georgas James , Mylonas Kostas , Bafiti Tsabika , Poortinga Ype H. , Christakopoulou Sophia , Kagitcibasi Cigdem , Kwak Kyunghwa , Ataca Bilge , Berry John , Orung Sabiha , Sunar Diane , Charalambous Neophytos , Goodwin Robin , Wang Wen‐Zhong , Angleitner Alois , Stepanikova Irena , Pick Susan , Givaudan Martha , Zhuravliova‐Gionis Irina , Konantambigi Rajani , Gelfand Michele J. , Marinova Velislava , McBride‐Chang Catherine , Kodiç Yasmin
Published: July 2002

This study investigated the relationship between culture, structural aspects of the nuclear and extended family, and functional aspects of the family, that is, emotional distance, social interaction, and communication, as well as geographical proximity. The focus was on the functional aspects of family, defined as members of the nuclear family (mother, father, and their children) and the extended family (grandmother/grandfather, aunt/uncle, cousins). Sixteen cultures participated in this study, with a total number of 2587 participants. The first hypothesis, that the pattern of scores on the psychological measures and the behavioral outcomes are similar across cultures, an indication of cultural universality, was supported. The second hypothesis, that functional relations between members of the nuclear family and their kin are maintained in high‐affluent and low‐affluent cultures, and that differences in functional relationships in high‐ and low‐affluent cultures are a matter of degree, was also supported by the findings. The results suggest that it is less meaningful in cross‐cultural family studies to ask questions about the structure of the family, than to ask about the functional relationships between members of the nuclear family and their kin. In looking only at the nuclear family, one focuses only on those residing in the household, but ignores those important members of the extended family who may reside nearby and their significant relationships with the members of the nuclear family.
La théorie de la privation relative et les réactions au handicap: Le rôle des comparaisons intrapersonnelles dans la gestion de l'estime de soi
Authors: Dif Stéphane , Guimond Serge , Martinot Delphine , Redersdorff Sandrine
Published: July 2002

La théorie de la privation relative (PR) distingue habituellement les comparaisons interpersonnelles des comparaisons intergroupes, mais néglige les comparaisons intrapersonnelles. Cette recherche révèle que les comparaisons entre le soi présent et le soi futur aboutissent à des sentiments de gratification relative (GR), eux‐mêmes reliés à l'estime de soi. Une première étude (N = 581) confirme que la GR intrapersonnelle contribue positivement à l'estime de soi. Une seconde étude, menée auprès d'étudiants handicapés (n = 31) et valides (n = 50) révèle que si les handicapés ont une estime de soi plus élevée que les valides, cette préservation s'explique uniquement par la GR intrapersonnelle. Enfin, la PR intergroupe et la GR intrapersonelle sont deux prédicteurs distincts du militantisme en faveur des personnes handicapées. Ces résultats attestent l'importance de différencier les niveaux de comparaisons.
Multipotential Abilities and Vocational Interests in Gifted Adolescents: Fact or Fiction?
Authors: Milgram Roberta M. , Hong Eunsook
Published: July 2002

The widely held view that highly intellectually gifted adolescents are multipotential in their abilities and career interests was investigated in two samples (N = 322 and N = 217) of gifted high school seniors. Three measures of intellectual abilities—verbal, mathematical, and mechanical—and a measure of vocational interest based upon nonacademic talented activities and accomplishments that yielded scores for vocational interest in three areas—Science, Social, and Artistic—were administered. Contrary to the widely held view, a very large proportion of intellectually gifted adolescents reported a differentiated pattern of both abilities and interests, and a very small proportion reported the expected high‐flat ability and interest profiles. The findings suggest that the working assumptions of career education and counselling of intellectually gifted adolescents should perhaps be reconsidered.
Industrial Clusters: Complexes, Agglomeration and/or Social Networks?
Authors: Gordon Ian R. , McCann Philip
Published: July 2002

The concept of industrial clusters has attracted much attention during the past decade, both as descriptive of an increasingly important phenomenon and as a basis for effective public intervention in the economies of lagging city-regions. However, there is much ambiguity in the way in which this concept is used, presenting an obstacle both to empirical testing and to realistic assessments of policy relevance. In this paper, we distinguish three ideal-typical models of processes which may underlie spatial concentrations of related activities, with very different implications both in terms of relevant evidence and the scope for promotional policies. Survey data for the London conurbation are used to explore the relation between concentration and different forms of linkage, with results which point to the dominance of pure agglomeration effects in this context at least.