bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: A projected basis for immunotherapy
Authors: Le Fur Nathalie , Kelsall Stephen R. , Silvers Willys K. , Mintz Beatrice
Published: July 2002

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase–PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Δ1b and Δ1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Δ1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Δ1d reached 4.0% as compared with 0.8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Δ1b and Δ1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.
Loss of functional cell surface transforming growth factor β (TGF-β) type 1 receptor correlates with insensitivity to TGF-β in chronic lymphocytic leukemia
Authors: DeCoteau John F. , Knaus Petra I. , Yankelev Haya , Reis Marciano D. , Lowsky Robert , Lodish Harvey F. , Kadin Marshall E.
Published: July 2002

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries, and there is significant variability in survival within CLL clinical stages. Earlier studies showed that CLL cells produce and are usually growth inhibited by transforming growth factor β type 1 (TGF-β1), suggesting a mechanism for the clinically indolent course of most CLL. Here we studied the mechanism by which CLL cells from about one-third of the patients are insensitive to TGF-β1. Of the 13 patients studied, CLL cells isolated from the peripheral blood of 8 patients were sensitive to growth inhibition by TGF-β1, as determined by incorporation of tritiated thymidine, whereas those from 5 patients were completely resistant to TGF-β1. As judged by binding of radiolabeled TGF-β1 followed by cross-linking and immunoprecipitation with anti-receptor antisera, CLL cells sensitive to TGF-β1 exhibited normal cell surface expression of both types 1 and 2 TGF-β receptors. In contrast, all CLL cells resistant to TGF-β1 exhibited no detectable surface type I receptors able to bind TGF-β1, but normal expression of type II receptors. Both TGF-β1-sensitive and TGF-β1-resistant CLL cells contained normal amounts of both type 1 and type 2 receptor mRNAs. Specific loss of type 1 receptor expression represents a new mechanism by which cells acquire resistance to TGF-β1-mediated growth inhibition in the development and progression of human lymphoproliferative malignancies.
Virus dynamics and drug therapy
Authors: Bonhoeffer Sebastian , May Robert M. , Shaw George M. , Nowak Martin A.
Published: July 2002

The recent development of potent antiviral drugs not only has raised hopes for effective treatment of infections with HIV or the hepatitis B virus, but also has led to important quantitative insights into viral dynamicsin vivo. Interpretation of the experimental data depends upon mathematical models that describe the nonlinear interaction between virus and host cell populations. Here we discuss the emerging understanding of virus population dynamics, the role of the immune system in limiting virus abundance, the dynamics of viral drug resistance, and the question of whether virus infection can be eliminated from individual patients by drug treatment.
Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP
Authors: van Bergen Jeroen , Schoenberger Stephen P. , Verreck Frank , Amons Reinout , Offringa Rienk , Koning Frits
Published: July 2002

The HLA class II-associated invariant chain (Ii)-derived peptide (CLIP) occupies the peptide binding groove during assembly in the endoplasmic reticulum, travels with HLA class II to endosomal compartments, and is subsequently released to allow binding of antigenic peptides. We investigated whether the exchange of CLIP with a known T helper epitope at the DNA level would lead to efficient loading of this helper epitope onto HLA class II. For this purpose, a versatile Ii-encoding expression vector was created in which CLIP can be replaced with a helper epitope of choice. Upon supertransfection of HLA-DR1-transfected 293 cells with an Ii vector encoding a known T helper epitope (HA307–319), predominantly length variants of this epitope were detected in association with the HLA-DR1 molecules of these cells. Moreover, this transfectant was efficiently recognized by a peptide-specific T helper clone (HA1.7). The results suggest that this type of Ii vector can be used to create potent class II+cellular vaccines in which defined T cell epitopes are continuously synthesized.
Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment
Authors: Ditzel Henrik J. , Garrigues Ursula , Andersen Claus B. , Larsen Morten K. , Garrigues H. Jacques , Svejgaard Arne , Hellström Ingegerd , Hellström Karl Erik , Jensenius Jens C.
Published: July 2002

Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage display selection and the human Fab fragment was expressed in bacteria. Analysis by confocal laser scanning microscopy demonstrated that COU-1 bound in a uniform punctate pattern to the surface of viable carcinoma cells stained at 4°C, and binding increased significantly when cells were cultured on fibronectin, laminin, or collagen IV. In the case of fibronectin, COU-1 staining was particularly enhanced at intercellular junctions. When carcinoma cells were cultured with COU-1 at 37°C for 6 hr, the antibody was found in large perinuclear vesicles and the punctate surface staining was significantly reduced. Similar results were obtained using intact IgM COU-1 and the recombinant Fab fragment. Immunohistological studies indicated that COU-1, in contrast to murine monoclonal antibodies against normal cytokeratin 8 and 18, could differentiate between malignant and normal colon epithelia, and between colon cancer metastasis in the liver and surrounding normal hepatocytes. Within biopsies of malignant tissue, COU-1 exhibited membrane-associated staining of proliferating cells, while resting cells had a filamentous pattern. Thus, modified cytokeratin at the surface of carcinoma cells may represent a new target for immunoconjugates and may explain the promising results of the phase I/II clinical study.
Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells
Authors: Schnierle Barbara S. , Stitz Jörn , Bosch Valerie , Nocken Frank , Merget-Millitzer Heike , Engelstädter Martin , Kurth Reinhard , Groner Bernd , Cichutek Klaus
Published: July 2002

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4
+
T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV
env
-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4
+
cells during gene therapy of AIDS.
Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets
Authors: Möhle Robert , Green David , Moore Malcolm A. S. , Nachman Ralph L. , Rafii Shahin
Published: July 2002

We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes. In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors. Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF). Megakaryocytes (CD41a
+
) were generated by
ex vivo
expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads. Using reverse transcription–PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids). Large quantities of VEGF (>1 ng/10
6
cells/3 days) were detected in the supernatant of Dami cells,
ex vivo
-generated megakaryocytes, and CD41a
+
cells isolated from bone marrow. The constitutive secretion of VEGF by CD41a
+
cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin). Western blotting of heparin–Sepharose-enriched supernatant mainly detected the isoform VEGF
165
. In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes. Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min. We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner. Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells. VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis.
The cloud–ionosphere discharge: A newly observed thunderstorm phenomenon
Authors: Winckler John R.
Published: July 2002

This paper deals with a luminous electric discharge that forms in the mesospheric region between thundercloud tops and the ionosphere at 90-km altitude. These cloud–ionosphere discharges (CIs), following visual reports dating back to the 19th century, were finally imaged by a low-light TV camera as part of the “SKYFLASH” program at the University of Minnesota in 1989. Many observations were made by various groups in the period 1993–1996. The characteristics of CIs are that they have a wide range of sizes from a few kilometers up to 50 km horizontally; they extend from 40 km to nearly 90 km vertically, with an intense region near 60–70 km and streamers extending down toward cloud tops; the CIs are partly or entirely composed of vertical luminous filaments of kilometer size. The predominate color is red. The TV images show that the CIs usually have a duration less than one TV field (16.7 ms), but higher-speed photometric measurements show that they last about 3 ms, and are delayed 3 ms after an initiating cloud–ground lightning stroke; 95% of these initiating strokes are found to be “positive”—i.e., carry positive charges from clouds to ground. The preference for positive initiating strokes is not understood. Theories of the formation of CIs are briefly reviewed.
A versatile synthetic dimerizer for the regulation of protein–protein interactions
Authors: Amara Jane F. , Clackson Tim , Rivera Victor M. , Guo Tao , Keenan Terence , Natesan Sridaran , Pollock Roy , Yang Wu , Courage Nancy L. , Holt Dennis A. , Gilman Michael
Published: July 2002

The use of low molecular weight organic compounds to induce dimerization or oligomerization of engineered proteins has wide-ranging utility in biological research as well as in gene and cell therapies. Chemically induced dimerization can be used to activate intracellular signal transduction pathways or to control the activity of a bipartite transcription factor. Dimerizer systems based on the natural products cyclosporin, FK506, rapamycin, and coumermycin have been described. However, owing to the complexity of these compounds, adjusting their binding or pharmacological properties by chemical modification is difficult. We have investigated several families of readily prepared, totally synthetic, cell-permeable dimerizers composed of ligands for human FKBP12. These molecules have significantly reduced complexity and greater adaptability than natural product dimers. We report here the efficacies of several of these new synthetic compounds in regulating two types of protein dimerization events inside engineered cells—–induction of apoptosis through dimerization of engineered Fas proteins and regulation of transcription through dimerization of transcription factor fusion proteins. One dimerizer in particular, AP1510, proved to be exceptionally potent and versatile in all experimental contexts tested.
Congruences between modular forms: Raising the level and dropping Euler factors
Authors: Diamond Fred
Published: July 2002

We discuss the relationship among certain generalizations of
results of Hida, Ribet, and Wiles on congruences between modular forms.
Hida’s result accounts for congruences in terms of the value of an
L
-function, and Ribet’s result is related to the
behavior of the period that appears there. Wiles’ theory leads to a
class number formula relating the value of the
L
-function to the size of a Galois cohomology group. The
behavior of the period is used to deduce that a formula at
“nonminimal level” is obtained from one at “minimal level” by
dropping Euler factors from the
L
-function.
MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-β superfamily
Authors: Bootcov Michelle R. , Bauskin Asne R. , Valenzuela Stella M. , Moore Anthony G. , Bansal Mohinder , He Xiao Yan , Zhang Hong Ping , Donnellan Melissa , Mahler Stephen , Pryor Kimberley , Walsh Bradley J. , Nicholson Richard C. , Fairlie W. Douglas , Por Suzanne B. , Robbins Joan M. , Breit Samuel N.
Published: July 2002

Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor β (TGF-β) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1β, tumor necrosis factor α (TNF-α), interleukin 2, and macrophage colony-stimulating factor but not interferon γ, or lipopolysaccharide (LPS). Its expression is also increased by TGF-β. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein ofMr25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-α production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-β may serve to limit the later phases of macrophage activation.
Effective intercellular communication distances are determined by the relative time constants for cyto/chemokine secretion and diffusion
Authors: Francis Karl , Palsson Bernhard O.
Published: July 2002

A cell’s ability to effectively communicate with a neighboring cell is essential for tissue function and ultimately for the organism to which it belongs. One important mode of intercellular communication is the release of soluble cyto- and chemokines. Once secreted, these signaling molecules diffuse through the surrounding medium and eventually bind to neighboring cell’s receptors whereby the signal is received. This mode of communication is governed both by physicochemical transport processes and cellular secretion rates, which in turn are determined by genetic and biochemical processes. The characteristics of transport processes have been known for some time, and information on the genetic and biochemical determinants of cellular function is rapidly growing. Simultaneous quantitative analysis of the two is required to systematically evaluate the nature and limitations of intercellular signaling. The present study uses a solitary cell model to estimate effective communication distances over which a single cell can meaningfully propagate a soluble signal. The analysis reveals that: (i) this process is governed by a single, key, dimensionless group that is a ratio of biological parameters and physicochemical determinants; (ii) this ratio has a maximal value; (iii) for realistic values of the parameters contained in this dimensionless group, it is estimated that the domain that a single cell can effectively communicate in is ≈250 μm in size; and (iv) the communication within this domain takes place in 10–30 minutes. These results have fundamental implications for interpretation of organ physiology and for engineering tissue functionex vivo.
Gene transfer of Fas ligand induces tumor regression in vivo
Authors: Arai Hiroshi , Gordon David , Nabel Elizabeth G. , Nabel Gary J.
Published: July 2002

The Fas–Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growthin vivo. Although such inhibition is expected in Fas+tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas+Renca tumor by inducing cell death, whereas the elimination of Fas−CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas+cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.
Adenylyl cyclase 6 is selectively regulated by protein kinase A phosphorylation in a region involved in Gα
s
stimulation
Authors: Chen Yibang , Harry Anya , Li Jingrong , Smit Martine J. , Bai Xu , Magnusson Ronald , Pieroni Joseph P. , Weng Gezhi , Iyengar Ravi
Published: July 2002

Receptors activate adenylyl cyclases through the Gα
s
subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by Gα
s
. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of Gα
s
. Similar treatment of AC1 or AC2 did not affect Gα
s
stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of Gα
s
stimulation. A peptide encoding the region 660–682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of Gα
s
. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660–682 of AC6 is involved in regulation of signal transfer from Gα
s
. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.
Is blindsight like normal, near-threshold vision?
Authors: Azzopardi Paul , Cowey Alan
Published: July 2002

Blindsight is the rare and paradoxical ability of some human subjects with occipital lobe brain damage to discriminate unseen stimuli in their clinically blind field defects when forced-choice procedures are used, implying that lesions of striate cortex produce a sharp dissociation between visual performance and visual awareness. Skeptics have argued that this is no different from the behavior of normal subjects at the lower limits of conscious vision, at which such dissociations could arise trivially by using different response criteria during clinical and forced-choice tests. We tested this claim explicitly by measuring the sensitivity of a hemianopic patient independently of his response criterion in yes-no and forced-choice detection tasks with the same stimulus and found that, unlike normal controls, his sensitivity was significantly higher during the forced-choice task. Thus, the dissociation by which blindsight is defined is not simply due to a difference in the patients’ response bias between the two paradigms. This result implies that blindsight is unlike normal, near-threshold vision and that information about the stimulus is processed in blindsighted patients in an unusual way.
Meis1 and pKnox1 bind DNA cooperatively with Pbx1 utilizing an interaction surface disrupted in oncoprotein E2a-Pbx1
Authors: Knoepfler Paul S. , Calvo Katherine R. , Chen Haiming , Antonarakis Stylianos E. , Kamps Mark P.
Published: July 2002

E2a-Pbx1 is a chimeric transcription factor oncoprotein produced by the
t
(1;19) translocation in human pre-B cell leukemia. Class I Hox proteins bind DNA cooperatively with both Pbx proteins and oncoprotein E2a-Pbx1, suggesting that leukemogenesis by E2a-Pbx1 and Hox proteins may alter transcription of cellular genes regulated by Pbx–Hox motifs. Likewise, in murine myeloid leukemia, transcriptional coactivation of Meis1 with HoxA7/A9 suggests that Meis1–HoxA7/9 heterodimers may evoke aberrant gene transcription. Here, we demonstrate that both Meis1 and its relative, pKnox1, dimerize with Pbx1 on the same TGATTGAC motif selected by dimers of Pbx proteins and unidentified partner(s) in nuclear extracts, including those from
t
(1;19) pre-B cells. Outside their homeodomains, Meis1 and pKnox1 were highly conserved only in two motifs required for cooperativity with Pbx1. Like the unidentified endogenous partner(s), both Meis1 and pKnox1 failed to dimerize significantly with E2a-Pbx1. The Meis1/pKnox1-interaction domain in Pbx1 resided predominantly in a conserved N-terminal Pbx domain deleted in E2a-Pbx1. Thus, the leukemic potential of E2a-Pbx1 may require abrogation of its interaction with members of the Meis and pKnox families of transcription factors, permitting selective targeting of genes regulated by Pbx–Hox complexes. In addition, because most motifs bound by Pbx–Meis1/pKnox1 were not bound by Pbx1–Hox complexes, the leukemic potential of Meis1 in myeloid leukemias may involve shifting Pbx proteins from promoters containing Pbx–Hox motifs to those containing Pbx–Meis motifs.
Role of left inferior prefrontal cortex in retrieval of semantic knowledge: A reevaluation
Authors: Thompson-Schill Sharon L. , D’Esposito Mark , Aguirre Geoffrey K. , Farah Martha J.
Published: July 2002

A number of neuroimaging findings have been interpreted as evidence that the left inferior frontal gyrus (IFG) subserves retrieval of semantic knowledge. We provide a fundamentally different interpretation, that it is not retrieval of semantic knowledge
per se
that is associated with left IFG activity but rather selection of information among competing alternatives from semantic memory. Selection demands were varied across three semantic tasks in a single group of subjects. Functional magnetic resonance imaging signal in overlapping regions of left IFG was dependent on selection demands in all three tasks. In addition, the degree of semantic processing was varied independently of selection demands in one of the tasks. The absence of left IFG activity for this comparison counters the argument that the effects of selection can be attributed solely to variations in degree of semantic retrieval. Our findings suggest that it is selection, not retrieval, of semantic knowledge that drives activity in the left IFG.
A single zinc finger motif in the silencing factor REST represses the neural-specific type II sodium channel promoter
Authors: Tapia-Ramírez José , Eggen Bart J. L. , Peral-Rubio Maria J. , Toledo-Aral Juan J. , Mandel Gail
Published: July 2002

The type II voltage-dependent sodium channel is present in neuronal cells, where it mediates the propagation of nerve impulses. Restricted expression of the type II sodium channel gene to neurons is due, at least in part, to binding of the repressor protein REST (also termed NRSF or XBR) to the RE1 (also called NRSE) sequence in the type II sodium channel gene. Previous studies have shown that a domain in REST containing eight GL1-Krüppel zinc finger motifs mediates DNA binding. Deletional and GAL4-fusion gene analyses now reveal repressor domains that lie outside of the DNA-binding domain in both the amino and carboxyl termini of REST. Mutational analysis further identifies a single zinc finger motif in the carboxyl-terminal domain as being essential for repressing type II sodium channel reporter genes. These studies reveal two domains in REST that may mediate interactions with other proteins involved in restricting expression of a large set of genes to the vertebrate nervous system.
Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine
Authors: Sparreboom Alex , van Asperen Judith , Mayer Ulrich , Schinkel Alfred H. , Smit Johan W. , Meijer Dirk K. F. , Borst Piet , Nooijen Willem J. , Beijnen Jos H. , van Tellingen Olaf
Published: July 2002

In mice, the
mdr1a
and
mdr1b
genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because
mdr1b
P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted
mdr1a
gene [
mdr1a
(−/−) mice] do not contain functional P-glycoprotein in this organ. We have used these
mdr1a
(−/−) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2- and 6-fold higher in
mdr1a
(−/−) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in
mdr1a
(−/−) mice. The cumulative fecal excretion (0–96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in
mdr1a
(−/−) mice. Biliary excretion was not significantly different in wt and
mdr1a
(−/−) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg/kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in
mdr1a
(−/−) mice. We conclude that P-glycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen.
Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution
Authors: Li Minglin , Liu Xiuwen , Robinson Gertraud , Bar-Peled Ud , Wagner Kay-Uwe , Young W. Scott , Hennighausen Lothar , Furth Priscilla A.
Published: July 2002

Programmed cell death (PCD) of mammary alveolar cells during involution commences within hours of the end of suckling. Locally, milk accumulates within alveolar lumens; systemically, levels of lactogenic hormones fall. Four experimental models were used to define the role of local factors as compared with systemic hormones during the first and second stages of involution. In three models, milk release was disrupted in the presence of systemic lactogenic hormones: (
i
) sealing of the teats, (
ii
) mammary gland transplants that cannot release milk due to the absence of a teat connection, and (
iii
) inactivation of the oxytocin gene. The ability of systemic hormones to preserve lobular-alveolar structure without blocking PCD was illustrated using a fourth transgenic model of lactation failure. During the first stage of involution, local signals were sufficient to induce alveolar PCD even in the presence of systemic lactogenic hormones. PCD coincided with bax induction, decreased expression of milk proteins, block of prolactin signal transduction through Stat5a and 5b, and activation of Stat3. The two stages of mammary gland involution are regulated by progressive gain of death signals and loss of survival factors. This study demonstrates that genetic events that occur during the first reversible stage are controlled by local factors. These mammary-derived death signals are dominant over protective effects related to systemic hormone stimulation.
DNA oxidation matters: The HPLC–electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine
Authors: Helbock Harold J. , Beckman Kenneth B. , Shigenaga Mark K. , Walter Patrick B. , Woodall Alan A. , Yeo Helen C. , Ames Bruce N.
Published: July 2002

Oxidative DNA damage is important in aging and the degenerative diseases of aging such as cancer. Estimates commonly rely on measurements of 8-oxo-2′-deoxyguanosine (oxo
8
dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair. Here we examine difficulties inherent in the analysis of oxo
8
dG, identify sources of artifacts, and provide solutions to some of the common methodological problems. A frequent criticism has been that phenol in DNA extraction solutions artificially increases the measured level of oxo
8
dG. We found that phenol extraction of DNA contributes a real but minor increase in the level of oxo
8
dG when compared, under equivalent conditions, with a successful nonphenol method. A more significant reduction in the baseline level was achieved with a modification of the recently introduced chaotropic NaI method, reducing our estimate of the level of steady-state oxidative adducts by an order of magnitude to 24,000 adducts per cell in young rats and 66,000 adducts per cell in old rats. Of several alternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo
8
dG values. In further studies we show that human urinary 8-oxo-guanine (oxo
8
Gua) excretion is not affected by the administration of allopurinol, suggesting that, unlike some methylated adducts, oxo
8
Gua is not derived enzymatically from xanthine oxidase. Lastly, we discuss remaining uncertainties inherent both in steady-state oxo
8
dG measurements and in estimates of endogenous oxidation (“hit rates”) based on urinary excretion of oxo
8
dG and oxo
8
Gua.
Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice
Authors: Horton Jay D. , Bashmakov Yuriy , Shimomura Iichiro , Shimano Hitoshi
Published: July 2002

Hepatic lipid synthesis is known to be regulated by food consumption. In rodents fasting decreases the synthesis of cholesterol as well as fatty acids. Refeeding a high carbohydrate/low fat diet enhances fatty acid synthesis by 5- to 20-fold above the fed state, whereas cholesterol synthesis returns only to the prefasted level. Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate genes involved in cholesterol and fatty acid synthesis. Here, we show that fasting markedly reduces the amounts of SREBP-1 and -2 in mouse liver nuclei, with corresponding decreases in the mRNAs for SREBP-activated target genes. Refeeding a high carbohydrate/low fat diet resulted in a 4- to 5-fold increase of nuclear SREBP-1 above nonfasted levels, whereas nuclear SREBP-2 protein returned only to the nonfasted level. The hepatic mRNAs for fatty acid biosynthetic enzymes increased 5- to 10-fold above nonfasted levels, a pattern that paralleled the changes in nuclear SREBP-1. The hepatic mRNAs for enzymes involved in cholesterol synthesis returned to the nonfasted level, closely following the pattern of nuclear SREBP-2 regulation. Transgenic mice that overproduce nuclear SREBP-1c failed to show the normal decrease in hepatic mRNA levels for cholesterol and fatty acid synthetic enzymes upon fasting. We conclude that SREBPs are regulated by food consumption in the mouse liver and that the decline in nuclear SREBP-1c upon fasting may explain in part the decrease in mRNAs encoding enzymes of the fatty acid biosynthetic pathway.
Secondary absence of mitochondria inGiardia lambliaandTrichomonas vaginalisrevealed by valyl-tRNA synthetase phylogeny
Authors: Hashimoto Tetsuo , Sánchez Lidya B. , Shirakura Tetsurou , Müller Miklós , Hasegawa Masami
Published: July 2002

Nuclear-coded valyl-tRNA synthetase (ValRS) of eukaryotes is regarded of mitochondrial origin. Complete ValRS sequences obtained by us from two amitochondriate protists, the diplomonad,Giardia lambliaand the parabasalid,Trichomonas vaginaliswere of the eukaryotic type, strongly suggesting an identical history of ValRS in all eukaryotes studied so far. The findings indicate that diplomonads are secondarily amitochondriate and give further evidence for such conclusion reached recently concerning parabasalids. Together with similar findings on other amitochondriate groups (microsporidia and entamoebids), this work provides critical support for the emerging notion that no representatives of the premitochondrial stage of eukaryotic phylogenesis exist among the species living today.
Demonstration of an interferon γ-dependent tumor surveillance system in immunocompetent mice
Authors: Kaplan Daniel H. , Shankaran Vijay , Dighe Anand S. , Stockert Elisabeth , Aguet Michel , Old Lloyd J. , Schreiber Robert D.
Published: July 2002

This study demonstrates that endogenously produced interferon γ (IFN-γ) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice. Compared with wild-type mice, mice lacking sensitivity to either IFN-γ (i.e., IFN-γ receptor-deficient mice) or all IFN family members (i.e., Stat1-deficient mice) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen methylcholanthrene. In addition, IFN-γ-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene. IFN-γ-insensitive p53−/−mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. Using tumor cells derived from methylcholanthrene-treated IFN-γ-insensitive mice, we found IFN-γ’s actions to be mediated at least partly through its direct effects on the tumor cell leading to enhanced tumor cell immunogenicity. The importance and generality of this system is evidenced by the finding that certain types of human tumors become selectively unresponsive to IFN-γ. Thus, IFN-γ forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts.
Evolution of the avian sex chromosomes from an ancestral pair of autosomes
Authors: Fridolfsson Anna-Karin , Cheng Hans , Copeland Neal G. , Jenkins Nancy A. , Liu Hsiao-Ching , Raudsepp Terje , Woodage Trevor , Chowdhary Bhanu , Halverson Joy , Ellegren Hans
Published: July 2002

Among the mechanisms whereby sex is determined in animals, chromosomal sex determination is found in a wide variety of distant taxa. The widespread but not ubiquitous occurrence, not even within lineages, of chromosomal sex determination suggests that sex chromosomes have evolved independently several times during animal radiation, but firm evidence for this is lacking. The most favored model for this process is gradual differentiation of ancestral pairs of autosomes. As known for mammals, sex chromosomes may have a very ancient origin, and it has even been speculated that the sex chromosomes of mammals and birds would share a common chromosomal ancestry. In this study we showed that the two genes,
ATP5A1
and
CHD1
, so far assigned to the female-specific W chromosome of birds both exist in a very closely related copy on the Z chromosome but are not pseudoautosomal. This indicates a common ancestry of the two sex chromosomes, consistent with the evolution from a pair of autosomes. Comparative mapping demonstrates, however, that
ATP5A1
and
CHD1
are not sex-linked among eutherian mammals; this is also not the case for the majority of other genes so far assigned to the avian Z chromosome. Our results suggest that the evolution of sex chromosomes has occurred independently in mammals and birds.
A post-recruitment function for the RNA polymerase III transcription–initiation factor IIIB
Authors: Kassavetis George A. , Kumar Ashok , Letts Garth A. , Geiduschek E. Peter
Published: July 2002

Transcription factor (TF) IIIB, which directs RNA polymerase (pol) III to its promoters, is made up of three components: the TATA box-binding protein, the TFIIB-related Brf, and the pol III-specific B′′. Certain mutations in
Saccharomyces cerevisiae
Brf and B′′ retain TFIIIB transcription factor activity with supercoiled DNA but are inactive with linear duplex DNA. Further analysis shows that these inactive TFIIIB–DNA complexes bind pol III and position it appropriately over the transcriptional start site but do not form DNA strand-separated open promoter complexes. It is proposed that the normal function of TFIIIB combines pol III recruitment with an active role in a subsequent step of transcriptional initiation leading to promoter opening.
P-selectin deficiency attenuates tumor growth and metastasis
Authors: Kim Young J. , Borsig Lubor , Varki Nissi M. , Varki Ajit
Published: July 2002

Selectins are adhesion receptors that normally recognize certain vascular mucin-type glycoproteins bearing the carbohydrate structure sialyl-Lewisx. The clinical prognosis and metastatic progression of many epithelial carcinomas has been correlated independently with production of tumor mucins and with enhanced expression of sialyl-Lewisx. Metastasis is thought to involve the formation of tumor-platelet-leukocyte emboli and their interactions with the endothelium of distant organs. We provide a link between these observations by showing that P-selectin, which normally binds leukocyte ligands, can promote tumor growth and facilitate the metastatic seeding of a mucin-producing carcinoma. P-selectin-deficient mice showed significantly slower growth of subcutaneously implanted human colon carcinoma cells and generated fewer lung metastases from intravenously injected cells. Three potential pathophysiological mechanisms are demonstrated: first, intravenously injected tumor cells home to the lungs of P-selectin deficient mice at a lower rate; second, P-selectin-deficient mouse platelets fail to adhere to tumor cell-surface mucins; and third, tumor cells lodged in lung vasculature after intravenous injection often are decorated with platelet clumps, and these are markedly diminished in P-selectin-deficient animals.
Epstein–Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-κB through a pathway that includes the NF-κB-inducing kinase and the IκB kinases IKKα and IKKβ
Authors: Sylla Bakary S. , Hung Siu Chun , Davidson David M. , Hatzivassiliou Eudoxia , Malinin Nikolai L. , Wallach David , Gilmore Thomas D. , Kieff Elliott , Mosialos George
Published: July 2002

The Epstein–Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively aggregated pseudo-tumor necrosis factor receptor (TNFR) that activates transcription factor NF-κB through two sites in its C-terminal cytoplasmic domain. One site is similar to activated TNFRII in associating with TNFR-associated factors TRAF1 and TRAF2, and the second site is similar to TNFRI in associating with the TNFRI death domain interacting protein TRADD. TNFRI has been recently shown to activate NF-κB through association with TRADD, RIP, and TRAF2; activation of the NF-κB-inducing kinase (NIK); activation of the IκBα kinases (IKKα and IKKβ); and phosphorylation of IκBα. IκBα phosphorylation on Ser-32 and Ser-36 is followed by its degradation and NF-κB activation. In this report, we show that NF-κB activation by LMP1 or by each of its effector sites is mediated by a pathway that includes NIK, IKKα, and IKKβ. Dominant negative mutants of NIK, IKKα, or IKKβ substantially inhibited NF-κB activation by LMP1 or by each of its effector sites.
Glucose and ethylene signal transduction crosstalk revealed by an
Arabidopsis
glucose-insensitive mutant
Authors: Zhou Li , Jang Jyan-chyun , Jones Tamara L. , Sheen Jen
Published: July 2002

Glucose is an essential signaling molecule that controls plant development and gene expression through largely unknown mechanisms. To initiate the dissection of the glucose signal transduction pathway in plants by using a genetic approach, we have identified an
Arabidopsis
mutant,
gin1
(glucose-insensitive), in which glucose repression of cotyledon greening and expansion, shoot development, floral transition, and gene expression is impaired. Genetic analysis indicates that
GIN1
acts downstream of the sensor hexokinase in the glucose signaling pathway. Surprisingly,
gin1
insensitivity to glucose repression of cotyledon and shoot development is phenocopied by ethylene precursor treatment of wild-type plants or by constitutive ethylene biosynthesis and constitutive ethylene signaling mutants. In contrast, the ethylene insensitive mutant
etr1-1
exhibits glucose hypersensitivity. Epistasis analysis places
GIN1
downstream of the ethylene receptor,
ETR1
, and defines a new branch of ethylene signaling pathway that is uncoupled from the triple response induced by ethylene. The isolation and characterization of
gin1
reveal an unexpected convergence between the glucose and the ethylene signal transduction pathways.
GIN1
may function to balance the control of plant development in response to metabolic and hormonal stimuli that act antagonistically.
Climate forcings in the Industrial era
Authors: Hansen James E. , Sato Makiko , Lacis Andrew , Ruedy Reto , Tegen Ina , Matthews Elaine
Published: July 2002

The forcings that drive long-term climate change are not known with
an accuracy sufficient to define future climate change. Anthropogenic
greenhouse gases (GHGs), which are well measured, cause a strong
positive (warming) forcing. But other, poorly measured, anthropogenic
forcings, especially changes of atmospheric aerosols, clouds, and
land-use patterns, cause a negative forcing that tends to offset
greenhouse warming. One consequence of this partial balance is that the
natural forcing due to solar irradiance changes may play a larger role
in long-term climate change than inferred from comparison with GHGs
alone. Current trends in GHG climate forcings are smaller than in
popular “business as usual” or 1% per year CO
2
growth scenarios. The summary implication is a paradigm change for
long-term climate projections: uncertainties in climate forcings have
supplanted global climate sensitivity as the predominant issue.
Cluster analysis and display of genome-wide expression patterns
Authors: Eisen Michael B. , Spellman Paul T. , Brown Patrick O. , Botstein David
Published: July 2002

A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast
Saccharomyces cerevisiae
that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.