bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.
Authors: Habraken Y , Sung P , Prakash S , Prakash L
Published: July 2002

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.
Two highly homologous ribonuclease genes expressed in mouse eosinophils identify a larger subgroup of the mammalian ribonuclease superfamily.
Authors: Larson K A , Olson E V , Madden B J , Gleich G J , Lee N A , Lee J J
Published: July 2002

Two putative ribonucleases have been isolated from the secondary granules of mouse eosinophils. Degenerate oligonucleotide primers inferred from peptide sequence data were used in reverse transcriptase-PCR reactions of bone marrow-derived cDNA. The resulting PCR product was used to screen a C57BL/6J bone marrow cDNA library, and comparisons of representative clones showed that these genes and encoded proteins are highly homologous (96% identity at the nucleotide level; 92/94% identical/similar at the amino acid level). The mouse proteins are only weakly homologous (approximately 50% amino acid identity) with the human eosinophil-associated ribonucleases (i.e., eosinophil-derived neurotoxin and eosinophil cationic protein) and show no sequence bias toward either human protein. Phylogenetic analyses established that the human and mouse loci shared an ancestral gene, but that independent duplication events have occurred since the divergence of primates and rodents. The duplication event generating the mouse genes was estimated to have occurred < 5 x 10(6) years ago (versus 30 to 40 x 10(6) years ago in primates). The identification of independent duplication events in two extant mammalian orders suggests a selective advantage to having multiple eosinophil granule ribonucleases. Southern blot analyses in the mouse demonstrated the existence of three additional highly homologous genes (i.e., five genes total) as well as several more divergent family members. The potential significance of this observation is the implication of a larger gene subfamily in primates (i.e., humans).
Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide dismutase
Authors: Emerit Ingrid , Garban Frédéric , Vassy Jany , Levy Arlette , Filipe Paulo , Freitas Joao
Published: July 2002

Superoxide-mediated clastogenesis is characteristic for various
chronic inflammatory diseases with autoimmune reactions and probably
plays a role in radiation-induced clastogenesis and in the congenital
breakage syndromes. It is consistently prevented by exogenous
superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating
that the anticlastogenic effect is related to the catalytic function of
the enzyme. Increased superoxide production by activated
monocytes/macrophages is followed by release of more long-lived
metabolites, so-called clastogenic factors, which contain lipid
peroxidation products, unusual nucleotides of inosine, and cytokines
such as tumor necrosis factor α. Since these components are not only
clastogenic, but can stimulate further superoxide production by
monocytes and neutrophils, the genotoxic effects are self-sustaining.
It is shown here that anticlastogenic effects of exogenous SOD are
preserved despite extensive washing of the cells and removal of all
extracellular SOD. Using flow cytometry and confocal laser microscopy,
rapid adherence of the fluorescently labeled enzyme to the cell surface
could be observed with slow uptake into the cell during the following
hours. The degree of labeling was concentration and time dependent. It
was most important for monocytes, compared with lymphocytes,
neutrophils, and fibroblasts. The cytochrome
c
assay
showed significantly diminished O
2
−
production by
monocytes, pretreated with SOD and washed thereafter. The preferential
and rapid binding of SOD to monocytes may be of importance not
only for the superoxide-mediated genotoxic effects, described above,
but also from a therapeutic standpoint. It can explain the observation
that beneficial effects of injected SOD lasted for weeks and months
despite rapid clearance of the enzyme from the blood stream according
to pharmacodynamic studies.
Activation of protein kinase A-independent pathways by G
s
α in
Drosophila
Authors: Wolfgang William J. , Roberts Ian J. H. , Quan Frank , O’Kane Cahir , Forte Michael
Published: July 2002

One of the best-described transmembrane signal transduction
mechanisms is based on receptor activation of the α subunit of the
heterotrimeric G protein G
s
, leading to stimulation of
adenylyl cyclase and the production of cAMP. Intracellular cAMP is then
thought to mediate its effects largely, if not entirely, by activation
of protein kinase A and the subsequent phosphorylation of substrates
which in turn control diverse cellular phenomena. In this report we
demonstrate, by two different methods, that reduction or elimination of
protein kinase A activity had no effect on phenotypes generated by
activation of G
s
α pathways in
Drosophila
wing epithelial cells. These genetic studies show that the
G
s
α pathway mediates its primary effects by a novel
pathway in differentiating wing epithelial cells. This novel pathway
may in part be responsible for some of the complex, cell-specific
responses observed following activation of this pathway in different
cell types.
New infinite families of exact sums of squares formulas, Jacobi elliptic functions, and Ramanujan’s tau function
Authors: Milne Stephen C.
Published: July 2002

In this paper, we give two infinite families of explicit exact formulas that generalize Jacobi’s (1829) 4 and 8 squares identities to 4n2or 4n(n+ 1) squares, respectively, without using cusp forms. Our 24 squares identity leads to a different formula for Ramanujan’s tau function τ(n), whennis odd. These results arise in the setting of Jacobi elliptic functions, Jacobi continued fractions, Hankel or Turánian determinants, Fourier series, Lambert series, inclusion/exclusion, Laplace expansion formula for determinants, and Schur functions. We have also obtained many additional infinite families of identities in this same setting that are analogous to the η-function identities in appendix I of Macdonald’s work [Macdonald, I. G. (1972)Invent. Math.15, 91–143]. A special case of our methods yields a proof of the two conjectured [Kac, V. G. and Wakimoto, M. (1994) inProgress in Mathematics, eds. Brylinski, J.-L., Brylinski, R., Guillemin, V. & Kac, V. (Birkhäuser Boston, Boston, MA), Vol. 123, pp. 415–456] identities involving representing a positive integer by sums of 4n2or 4n(n+ 1) triangular numbers, respectively. Our 16 and 24 squares identities were originally obtained via multiple basic hypergeometric series, Gustafson’sCℓnonterminating6φ5summation theorem, and Andrews’ basic hypergeometric series proof of Jacobi’s 4 and 8 squares identities. We have (elsewhere) applied symmetry and Schur function techniques to this original approach to prove the existence of similar infinite families of sums of squares identities forn2orn(n+ 1) squares, respectively. Our sums of more than 8 squares identities are not the same as the formulas of Mathews (1895), Glaisher (1907), Ramanujan (1916), Mordell (1917, 1919), Hardy (1918, 1920), Kac and Wakimoto, and many others.
GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of Gα
i
subunits
Authors: De Vries Luc , Elenko Eric , Hubler Laura , Jones Teresa L. Z. , Farquhar Marilyn Gist
Published: July 2002

GAIP (G Alpha Interacting Protein) is a member of the recently
described RGS (Regulators of G-protein Signaling) family that was
isolated by interaction cloning with the heterotrimeric G-protein
Gα
i3
and was recently shown to be a GTPase-activating
protein (GAP). In AtT-20 cells stably expressing GAIP, we found that
GAIP is membrane-anchored and faces the cytoplasm, because it was not
released by sodium carbonate treatment but was digested by proteinase
K. When Cos cells were transiently transfected with GAIP and
metabolically labeled with [
35
S]methionine, two pools of
GAIP—a soluble and a membrane-anchored pool—were found. Since the N
terminus of GAIP contains a cysteine string motif and cysteine string
proteins are heavily palmitoylated, we investigated the possibility
that membrane-anchored GAIP might be palmitoylated. We found that after
labeling with [
3
H]palmitic acid, the membrane-anchored
pool but not the soluble pool was palmitoylated. In the yeast
two-hybrid system, GAIP was found to interact specifically with members
of the Gα
i
subfamily, Gα
i1
,
Gα
i2
, Gα
i3
, Gα
z
, and
Gα
o
, but not with members of other Gα subfamilies,
Gα
s
, Gα
q
, and Gα
12/13
. The C
terminus of Gα
i3
is important for binding because a 10-aa
C-terminal truncation and a point mutant of Gα
i3
showed
significantly diminished interaction. GAIP interacted preferentially
with the activated (GTP) form of Gα
i3
, which is in
keeping with its GAP activity. We conclude that GAIP is a
membrane-anchored GAP with a cysteine string motif. This motif, present
in cysteine string proteins found on synaptic vesicles, pancreatic
zymogen granules, and chromaffin granules, suggests GAIP’s possible
involvement in membrane trafficking.
Retinoid X receptor-selective ligands produce malformations in Xenopus embryos.
Authors: Minucci S , Saint-Jeannet J P , Toyama R , Scita G , DeLuca L M , Tiara M , Levin A A , Ozato K , Dawid I B
Published: July 2002

Retinoids exert pleiotropic effects on the development of vertebrates through the action of retinoic acid receptors (RAR) and retinoid X receptors (RXR). We have investigated the effect of synthetic retinoids selective for RXR and RAR on the development of Xenopus and zebrafish embryos. In Xenopus, both ligands selective for RAR and RXR caused striking malformations along the anterior-posterior axis, whereas in zebrafish only ligands specific for RAR caused embryonic malformations. In Xenopus, RAR- and RXR-selective ligands regulated the expression of the Xlim-1, gsc, and HoxA1 genes similarly as all-trans-retinoic acid. Nevertheless, RXR-selective ligands activated only an RXR responsive reporter but not an RAR responsive reporter introduced by microinjection into the Xenopus embryo, consistent with our failure to detect conversion of an RXR-selective ligand to different derivatives in the embryo. These results suggest that Xenopus embryos possess a unique response pathway in which liganded RXR can control gene expression. Our observations further illustrate the divergence in retinoid responsiveness between different vertebrate species.
Crystal structure of the T state of allosteric yeast chorismate mutase and comparison with the R state.
Authors: Strater N , Hakansson K , Schnappauf G , Braus G , Lipscomb W N
Published: July 2002

The crystal structure of the tyrosine-bound T state of allosteric yeast Saccharomyces cerevisiae chorismate mutase was solved by molecular replacement at a resolution of 2.8 angstroms using a monomer of the R-state structure as the search model. The allosteric inhibitor tyrosine was found to bind in the T state at the same binding site as the allosteric activator tryptophan binds in the R state, thus defining one regulatory binding site for each monomer. Activation by tryptophan is caused by the larger steric size of its side chain, thereby pushing apart the allosteric domain of one monomer and helix H8 of the catalytic domain of the other monomer. Inhibition is caused by polar contacts of tyrosine with Arg-75 and Arg-76 of one monomer and with Gly-141, Ser-142, and Thr-145 of the other monomer, thereby bringing the allosteric and catalytic domains closer together. The allosteric transition includes an 8 degree rotation of each of the two catalytic domains relative to the allosteric domains of each monomer (domain closure). Alternatively, this transition can be described as a 15 degree rotation of the catalytic domains of the dimer relative to each other.
Hypothesis testing and earthquake prediction.
Authors: Jackson D D
Published: July 2002

Requirements for testing include advance specification of the conditional rate density (probability per unit time, area, and magnitude) or, alternatively, probabilities for specified intervals of time, space, and magnitude. Here I consider testing fully specified hypotheses, with no parameter adjustments or arbitrary decisions allowed during the test period. Because it may take decades to validate prediction methods, it is worthwhile to formulate testable hypotheses carefully in advance. Earthquake prediction generally implies that the probability will be temporarily higher than normal. Such a statement requires knowledge of "normal behavior"--that is, it requires a null hypothesis. Hypotheses can be tested in three ways: (i) by comparing the number of actual earth-quakes to the number predicted, (ii) by comparing the likelihood score of actual earthquakes to the predicted distribution, and (iii) by comparing the likelihood ratio to that of a null hypothesis. The first two tests are purely self-consistency tests, while the third is a direct comparison of two hypotheses. Predictions made without a statement of probability are very difficult to test, and any test must be based on the ratio of earthquakes in and out of the forecast regions.
The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored byin vitrophotorepair of cyclobutane pyrimidine dimers
Authors: Vink Arie A. , Moodycliffe Angus M. , Shreedhar Vijay , Ullrich Stephen E. , Roza Len , Yarosh Daniel B. , Kripke Margaret L.
Published: July 2002

Exposing skin to UVB (280–320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen afterin vivoUV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubatedin vitrowith liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored thein vivoantigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320–400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function.
Expression of a mutant α-zein creates the
floury
2 phenotype in transgenic maize
Authors: Coleman Craig E. , Clore Amy M. , Ranch Jerry P. , Higgins Regina , Lopes Mauricio A. , Larkins Brian A.
Published: July 2002

The maize
floury2
mutation results in the formation of a soft, starchy endosperm with a reduced amount of prolamin (zein) proteins and twice the lysine content of the wild type. The mutation is semidominant and is associated with small, irregularly shaped protein bodies, elevated levels of a 70-kDa chaperone in the endoplasmic reticulum, and a novel 24-kDa polypeptide in the zein fraction. The 24-kDa polypeptide is a precursor of a 22-kDa α-zein protein that is not properly processed. The defect is due to an alanine-to-valine substitution at the C-terminal position of the signal peptide, which causes the protein to be anchored to the endoplasmic reticulum. We postulated that the phenotype associated with the
floury2
mutation is caused by the accumulation of the 24-kDa α-zein protein. To test this hypothesis, we created transgenic maize plants that produce the mutant protein. We found that endosperm in seeds of these plants manifests the
floury2
phenotype, thereby confirming that the mutant α-zein is the molecular basis of this mutation.
Chemical camouflage of antigenic determinants: Stealth erythrocytes
Authors: Scott Mark D. , Murad Kari L. , Koumpouras Fotios , Talbot Margaret , Eaton John W.
Published: July 2002

In a number of clinical circumstances it would be desirable to artificially conceal cellular antigenic determinants to permit survival of heterologous donor cells. A case in point is the problem encountered in transfusions of patients with rare blood types or chronically transfused patients who become allosensitized to minor blood group determinants. We have tested the possibility that chemical modification of the red blood cell (RBC) membrane might serve to occlude antigenic determinants, thereby minimizing transfusion reactions. To this end, we have covalently bound methoxy(polyethylene glycol) (mPEG) to the surface of mammalian RBC via cyanuric chloride coupling. Human RBC treated with this technique lose ABO blood group reactivity as assessed by solution–phase antisera agglutination. In accord with this, we also find a profound decrease in anti-blood group antibody binding. Furthermore, whereas human monocytes avidly phagocytose untreated sheep RBC, mPEG-derivatized sheep RBC are ineffectively phagocytosed. Surprisingly, human and mouse RBC appear unaffected by this covalent modification of the cell membrane. Thus, mPEG-treated RBC are morphologically normal, have normal osmotic fragility, and mPEG-derivatized murine RBC have normal
in vivo
survival, even following repeated infusions. Finally, in preliminary experiments, mPEG-modified sheep RBC intraperitoneally transfused into mice show significantly improved (up to 360-fold) survival when compared with untreated sheep RBC. We speculate that similar chemical camouflage of intact cells may have significant clinical applications in both transfusion (e.g., allosensitization and autoimmune hemolytic disease) and transplantation (e.g., endothelial cells and pancreatic β cells) medicine.
Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype
Authors: Zhang Ning , Chen Philip , Khanna Kum Kum , Scott Shaun , Gatei Magtouf , Kozlov Sergei , Watters Dianne , Spring Kevin , Yen Tim , Lavin Martin F.
Published: July 2002

A gene mutated in the human genetic disorder ataxia-telangiectasia (A-T), ATM, was recently identified by positional cloning. ATM is a member of the phosphatidylinositol-3-kinase superfamily, some of which are protein kinases and appear to have important roles in cell cycle control and radiation signal transduction. We describe herein, to our knowledge, for the first time, the cloning of a full-length cDNA for ATM and correction of multiple aspects of the radio-sensitive phenotype of A-T cells by transfection with this cDNA. Overexpression of ATM cDNA in A-T cells enhanced the survival of these cells in response to radiation exposure, decreased radiation-induced chromosome aberrations, reduced radio-resistant DNA synthesis, and partially corrected defective cell cycle checkpoints and induction of stress-activated protein kinase. This correction of the defects in A-T cells provides further evidence of the multiplicity of effector functions of the ATM protein and suggests possible approaches to gene therapy.
Efficient gene tagging in
Arabidopsis thaliana
using a gene trap approach
Authors: Babiychuk Elena , Fuangthong Mayuree , Van Montagu Marc , Inzé Dirk , Kushnir Sergei
Published: July 2002

Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in
Arabidopsis thaliana
. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (
nptII
) depends on the
in vivo
generation of a translation fusion upon the T-DNA integration into the
Arabidopsis
genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for
Arabidopsis
. The results presented here suggest that gene trapping, using
nptII
as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in
A. thaliana
.
A link between protein structure and enzyme catalyzed hydrogen tunneling
Authors: Bahnson Brian J. , Colby Thomas D. , Chin Jodie K. , Goldstein Barry M. , Klinman Judith P.
Published: July 2002

We present evidence that the size of an active site side chain may
modulate the degree of hydrogen tunneling in an enzyme-catalyzed
reaction. Primary and secondary
k
H
/k
T
and
k
D
/k
T
kinetic
isotope effects have been measured for the oxidation of benzyl alcohol
catalyzed by horse liver alcohol dehydrogenase at 25°C. As reported
in earlier studies, the relationship between secondary
k
H
/k
T
and
k
D
/k
T
isotope
effects provides a sensitive probe for deviations from classical
behavior. In the present work, catalytic efficiency and the extent of
hydrogen tunneling have been correlated for the alcohol
dehydrogenase-catalyzed hydride transfer among a group of site-directed
mutants at position 203. Val-203 interacts with the opposite face of
the cofactor NAD
+
from the alcohol substrate. The reduction
in size of this residue is correlated with diminished tunneling and a
two orders of magnitude decrease in catalytic efficiency. Comparison of
the x-ray crystal structures of a ternary complex of a high-tunneling
(Phe-93 → Trp) and a low-tunneling (Val-203 → Ala) mutant provides
a structural basis for the observed effects, demonstrating an increase
in the hydrogen transfer distance for the low-tunneling mutant. The
Val-203 → Ala ternary complex crystal structure also shows a
hyperclosed interdomain geometry relative to the wild-type and the
Phe-93 → Trp mutant ternary complex structures. This demonstrates a
flexibility in interdomain movement that could potentially narrow the
distance between the donor and acceptor carbons in the native enzyme
and may enhance the role of tunneling in the hydride transfer reaction.
Natural killer cell lines kill autologous β2-microglobulin-deficient melanoma cells: Implications for cancer immunotherapy
Authors: Porgador Angel , Mandelboim Ofer , Restifo Nicholas P. , Strominger Jack L.
Published: July 2002

Cancer vaccines used to generate specific cytotoxic T lymphocytes are not effective against tumor cells that have lost or suppressed expression of their class I major histocompatibility complex proteins. This loss is common in some cancers and particularly in metastatic lesions. We show that β2-microglobulin-deficient class I-negative melanoma variants derived from patients undergoing specific T cell therapy are lysed by heterologous as well as autologous natural killer (NK) lines and clones, but not by specific T cells. Moreover, the minor NK cell fraction but not the major T cell fraction derived from heterologous lymphokine activated killer cells kills those tumor cell lines. ICAM-1 expression by the different class I protein deficient tumors was correlated with their sensitivity to lysis by NK cells. Adoptive autologous NK therapy may be an important supplement to consider in the design of new cancer immunotherapies.
Visualization of a peripheral stalk in V-type ATPase: Evidence for the stator structure essential to rotational catalysis
Authors: Boekema E. J. , Ubbink-Kok T. , Lolkema J. S. , Brisson A. , Konings W. N.
Published: July 2002

F- and V-type ATPases are central enzymes in energy metabolism that couple synthesis or hydrolysis of ATP to the translocation of H
+
or Na
+
across biological membranes. They consist of a soluble headpiece that contains the catalytic sites and an integral membrane-bound part that conducts the ion flow. Energy coupling is thought to occur through the physical rotation of a stalk that connects the two parts of the enzyme complex. This mechanism implies that a stator-like structure prevents the rotation of the headpiece relative to the membrane-bound part. Such a structure has not been observed to date. Here, we report the projected structure of the V-type Na
+
-ATPase of
Clostridium fervidus
as determined by electron microscopy. Besides the central stalk, a second stalk of 130 Å in length is observed that connects the headpiece and membrane-bound part in the periphery of the complex. This additional stalk is likely to be the stator.
Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways
Authors: Arbiser Jack L. , Moses Marsha A. , Fernandez Cecilia A. , Ghiso Neil , Cao Yihai , Klauber Nancy , Frank David , Brownlee Michael , Flynn Evelyn , Parangi Sareh , Byers H. Randolph , Folkman Judah
Published: July 2002

The switch from a quiescent tumor to an invasive tumor is accompanied by the acquisition of angiogenic properties. This phenotypic change likely requires a change in the balance of angiogenic stimulators and angiogenic inhibitors. The nature of the angiogenic switch is not known. Here, we show that introduction of activated H-ras into immortalized endothelial cells is capable of activating the angiogenic switch. Angiogenic switching is accompanied by up-regulation of vascular endothelial growth factor and matrix metalloproteinase (MMP) bioactivity and down-regulation of tissue inhibitor of MMP. Furthermore, we show that inhibition of phosphatidylinositol-3-kinase leads to partial inhibition of tumor angiogenesis, thus demonstrating that activated H-ras activates tumor angiogenesis through two distinct pathways. Finally, we show evidence for two forms of tumor dormancy.
Mutagenicity of 5-aza-2′-deoxycytidine is mediated by the mammalian DNA methyltransferase
Authors: Jackson-Grusby Laurie , Laird Peter W. , Magge Suresh N. , Moeller Benjamin J. , Jaenisch Rudolf
Published: July 2002

The cytosine analog 5-aza-2′-deoxycytidine has been used clinically to reactivate genes silenced by DNA methylation. In particular, patients with β-thalassemia show fetal globin expression after administration of this hypomethylating drug. In addition, silencing of tumor suppressor gene expression by aberrant DNA methylation in tumor cells may potentially be reversed by a similar regimen. Consistent with its function in maintaining tumor suppressor gene expression, 5-aza-2′-deoxycytidine significantly reduces intestinal tumor multiplicity in the predisposed Min mouse strain. Despite its utility as an anti-cancer agent, the drug is highly mutagenic by an unknown mechanism. To gain insight into how 5-aza-2′-deoxycytidine induces mutations
in vivo
, we examined the mutational spectrum in an
Escherichia coli lac I
transgene in colonic DNA from 5-aza-2′-deoxycytidine-treated mice. Mutations induced by 5-aza-2′-deoxycytidine were predominantly at CpG dinucleotides, which implicates DNA methyltransferase in the mutagenic mechanism. C:G→G:C transversions were the predominant class of mutations observed. We suggest a model for how the mammalian DNA methyltransferase may be involved in facilitating these mutations. The observation that 5-aza-2′-deoxycytidine-induced mutations are mediated by the enzyme suggests that novel inhibitors of DNA methyltransferase, which can inactivate the enzyme before its interaction with DNA, are needed for chemoprevention or long term therapy.
Sphingoid base 1-phosphate phosphatase: A key regulator of sphingolipid metabolism and stress response
Authors: Mandala Suzanne M. , Thornton Rosemary , Tu Zhenxing , Kurtz Myra B. , Nickels Joseph , Broach James , Menzeleev Ramil , Spiegel Sarah
Published: July 2002

The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that influences growth and survival of yeast. Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G
1
arrest and cell death. We now have identified a gene in
Saccharomyces cerevisiae, LBP1,
that regulates the levels of phosphorylated sphingoid bases and ceramide.
LBP1
was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis.
LBP1
and its homolog,
LBP2
, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity.
In vitro
characterization of Lbp1p shows that this phosphatase is Mg
2+
-independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine. The deletion of
LBP1
results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of
LBP1
and
LBP2
results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.
Isoform-specific effects of human apolipoprotein E on brain function revealed in
ApoE
knockout mice: Increased susceptibility of females
Authors: Raber Jacob , Wong Derek , Buttini Manuel , Orth Matthias , Bellosta Stefano , Pitas Robert E. , Mahley Robert W. , Mucke Lennart
Published: July 2002

Apolipoprotein E (apoE) mediates the redistribution of lipids among cells and is expressed at highest levels in brain and liver. Human apoE exists in three major isoforms encoded by distinct alleles (ɛ
2
, ɛ
3
, and ɛ
4
). Compared with APOE ɛ
2
and ɛ
3
, APOE ɛ
4
increases the risk of cognitive impairments, lowers the age of onset of Alzheimer’s disease (AD), and decreases the response to AD treatments. Besides age, inheritance of the APOE ɛ
4
allele is the most important known risk factor for the development of sporadic AD, the most common form of this illness. Although numerous hypotheses have been advanced, it remains unclear how APOE ɛ
4
might affect cognition and increase AD risk. To assess the effects of distinct human apoE isoforms on the brain, we have used the neuron-specific enolase (NSE) promoter to express human apoE3 or apoE4 at similar levels in neurons of transgenic mice lacking endogenous mouse apoE. Compared with NSE-apoE3 mice and wild-type controls, NSE-apoE4 mice showed impairments in learning a water maze task and in vertical exploratory behavior that increased with age and were seen primarily in females. These findings demonstrate that human apoE isoforms have differential effects on brain function
in vivo
and that the susceptibility to apoE4-induced deficits is critically influenced by age and gender. These results could be pertinent to cognitive impairments observed in human APOE ɛ
4
carriers. NSE-apoE mice and similar models may facilitate the preclinical assessment of treatments for apoE-related cognitive deficits.
The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation
Authors: Yamashita Takayuki , Kupfer Gary M. , Naf Dieter , Suliman Ahmed , Joenje Hans , Asano Shigetaka , D’Andrea Alan D.
Published: July 2002

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAA/FAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAA/FAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway.
Carcinogen-induced loss of heterozygosity at the
Aprt
locus in somatic cells of the mouse
Authors: Wijnhoven Susan W. P. , Van Sloun Petra P. H. , Kool Hanneke J. M. , Weeda Geert , Slater Rosalyn , Lohman Paul H. M. , van Zeeland Albert A. , Vrieling Harry
Published: July 2002

Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (
Aprt
) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH
in vivo
. After 7,12-dimethyl-1,2-benz[
a
]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal
Aprt
gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (
Hprt)
gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted
Aprt
allele was lost in 70% of the DMBA-induced
Aprt
mutants. Fluorescence
in situ
hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at
Aprt
. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss and duplication but not deletion. However, after treatment with the alkylating agent
N
-ethyl-
N
-nitrosourea,
Aprt
had a similar mutagenic response to
Hprt
while the majority (90%) of
N
-ethyl-
N
-nitrosourea-induced
Aprt
mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at
Hprt
but not at
Aprt
. This
in vivo
study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival and may represent an initiating event in tumorigenesis.
The adaptor protein Crk connects multiple cellular stimuli to the JNK signaling pathway
Authors: Dolfi Fabrizio , Garcia-Guzman Miguel , Ojaniemi Marja , Nakamura Hitoshi , Matsuda Michiyuki , Vuori Kristiina
Published: July 2002

c-Jun N-terminal kinases (JNKs) are potently activated by a number of cellular stimuli. Small GTPases, in particular Rac, are responsible for initiating the activation of the JNK pathways. So far, the signals leading from extracellular stimuli to the activation of Rac have remained elusive. Recent studies have demonstrated that the Src homology 2 (SH2)- and Src homology 3 (SH3)-containing adaptor protein Crk is capable of activating JNK when ectopically expressed. We found here that transient expression of Crk induces JNK activation, and this activation was dependent on both the SH2- and SH3-domains of Crk. Expression of p130
Cas
(Cas), a major binding protein for the Crk SH2-domain, also induced JNK activation, which was blocked by the SH2-mutant of Crk. JNK activation by Cas and Crk was effectively blocked by a dominant-negative form of Rac, suggesting for a linear pathway from the Cas-Crk-complex to the Rac-JNK activation. Many of the stimuli that activate the Rac-JNK pathway enhance engagement of the Crk SH2-domain. JNK activation by these stimuli, such as epidermal growth factor, integrin ligand binding and v-Src, was efficiently blocked by dominant-negative mutants of Crk. A dominant-negative form of Cas in turn blocked the integrin-, but not epidermal growth factor - nor v-Src-mediated JNK activation. Together, these results demonstrate an important role for Crk in connecting multiple cellular stimuli to the Rac-JNK pathway, and a role for the Cas-Crk complex in integrin-mediated JNK activation.
Exit of major histocompatibility complex class II–invariant chain p35 complexes from the endoplasmic reticulum is modulated by phosphorylation
Authors: Kuwana Tomomi , Peterson Per A. , Karlsson Lars
Published: July 2002

The Iip35 isoform of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains an endoplasmic reticulum (ER) targeting motif, but in B cell lines the ER retention is ineffective and a fraction of Iip35 is transported through the Golgi complex associated with class II molecules. We found Iip35 (but not Iip33, the major form of Ii) to be phosphorylated in B cell lines, as well as in transfected HeLa cells. The phosphorylation of Iip35 was found to be necessary for the exit of Iip35–class II complexes out of the ER. This requirement suggests that phosphorylation may change the interaction with factors responsible for ER retention/retrieval, and we did find that phosphorylated Iip35 associates with 14-3-3 proteins, a family of adaptor proteins that are involved in coordinating signal transduction pathways. This finding raises the intriguing possibility that the exit of Ii–class II complexes from the ER is regulated by intracellular signaling events.
Readily releasable pool size changes associated with long term depression
Authors: Goda Yukiko , Stevens Charles F.
Published: July 2002

We have estimated, for hippocampal neurons in culture, the size of the autaptic readily releasable pool before and after stimulation of the sort that produces culture long term depression (LTD). This stimulation protocol causes a decrease in the pool size that is proportional to the depression of synaptic currents. To determine if depression in this system is synapse specific rather than general, we have also monitored synaptic transmission between pairs of cultured hippocampal neurons that are autaptically and reciprocally interconnected. We find that the change in synaptic strength is restricted to the synapses on the target neuron that were active during LTD induction. When viewed from the perspective of the presynaptic neuron, however, synapse specificity is partial rather than complete: synapses active during induction that were not on the target neuron were partially depressed.
Inhibition of inositol 1,4,5-trisphosphate-induced Ca
2+
release by cAMP-dependent protein kinase in a living cell
Authors: Tertyshnikova Svetlana , Fein Alan
Published: July 2002

Interaction of intracellular free calcium ([Ca
2+
]
i
) and cAMP signaling mechanisms was examined in intact single megakaryocytes by using a combination of single-cell fluorescence microscopy to measure [Ca
2+
]
i
and flash photolysis of caged Ca
2+
, inositol 1,4,5-trisphosphate (IP
3
), or cAMP to elevate rapidly the concentration of these compounds inside the cell. Photolysis of caged IP
3
stimulated Ca
2+
release from an IP
3
-sensitive store. The cAMP-elevating agent carbacyclin inhibited this IP
3
-induced rise in [Ca
2+
]
i
but did not affect the rate of Ca
2+
removal from the cytoplasm after photolysis of caged Ca
2+
. Photolysis of caged cAMP during ADP-induced [Ca
2+
]
i
oscillations caused the [Ca
2+
]
i
oscillation to transiently cease without affecting the rate of Ca
2+
uptake and/or extrusion. We conclude that the principal mechanism of cAMP-dependent inhibition of Ca
2+
mobilization in megakaryocytes appears to be by inhibition of IP
3
-induced Ca
2+
release and not by stimulation of Ca
2+
removal from the cytoplasm. Two inhibitors of cAMP-dependent protein kinase, a specific peptide inhibitor of the catalytic subunit of cAMP protein kinase and KT5720, blocked the inhibitory effect of carbacyclin, indicating that the inhibition of IP
3
-induced Ca
2+
-release by carbacyclin is mediated by cAMP-dependent protein kinase.
Identification of a nucleotide binding site in HIV-1 integrase
Authors: Drake Richard R. , Neamati Nouri , Hong Huixiao , Pilon Andre A. , Sunthankar Prasanna , Hume Steven D. , Milne George W. A. , Pommier Yves
Published: July 2002

HIV-1 integrase is essential for viral replication and can be inhibited by antiviral nucleotides. Photoaffinity labeling with the 3′-azido-3′-deoxythymidine (AZT) analog 3′,5-diazido-2′,3′-dideoxyuridine 5′-monophosphate (5N
3
-AZTMP) and proteolytic mapping identified the amino acid 153–167 region of integrase as the site of photocrosslinking. Docking of 5N
3
-AZTMP revealed the possibility for strong hydrogen bonds between the inhibitor and lysines 156, 159, and 160 of the enzyme. Mutation of these residues reduced photocrosslinking selectively. This report elucidates the binding site of a nucleotide inhibitor of HIV-1 integrase, and possibly a component of the enzyme polynucleotide binding site.
The μ opiate receptor as a candidate gene for pain: Polymorphisms, variations in expression, nociception, and opiate responses
Authors: Uhl George R. , Sora Ichiro , Wang Zaijie
Published: July 2002

There are differences between human individuals and between mouse
strains in levels of μ opiate receptor (μOR) expression, responses
to painful stimuli, and responses to opiate drugs. One of the best
candidates for contributing to these differences is variation at the
μOR gene locus. Support for this idea comes from analyses of the
human and murine μOR genes. Assessments of individual differences in
human μOR expression add further support. Studies with mice,
including knockout-transgenic, quantitative trait locus, and
strain-comparison studies, also strongly support the possibility that
μOR gene alleles would be strong candidates for contributing to
individual differences in human nociception and opiate drug responses.
This paper reviews current analyses of the murine and human μOR
genes, their important variants, and correlations between these
variants and opiate influences on pain.
The mast cell tumor necrosis factor α response to FimH-expressingEscherichia coliis mediated by the glycosylphosphatidylinositol-anchored molecule CD48
Authors: Malaviya Ravi , Gao Zhimin , Thankavel Krishnan , van der Merwe P. Anton , Abraham Soman N.
Published: July 2002

Mast cells are well known for their harmful role in IgE-mediated hypersensitivity reactions, but their physiological role remains a mystery. Several recent studies have reported that mast cells play a critical role in innate immunity in mice by releasing tumor necrosis factor α (TNF-α) to recruit neutrophils to sites of enterobacterial infection. In some cases, the mast cell TNF-α response was triggered when these cells directly bound FimH on the surface ofEscherichia coli. We have identified CD48, a glycosylphosphatidylinositol-anchored molecule, to be the complementary FimH-binding moiety in rodent mast cell membrane fractions. We showed that (i) pretreatment of mast cell membranes with antibodies to CD48 or phospholipase C inhibited binding of FimH+E. coli, (ii) FimH+E. colibut not a FimH−derivative bound isolated CD48 in a mannose-inhibitable manner, (iii) binding of FimH+bacteria to Chinese hamster ovary (CHO) cells was markedly increased when these cells were transfected with CD48 cDNA, and (iv) antibodies to CD48 specifically blocked the mast cell TNF-α response to FimH+E. coli.Thus, CD48 is a functionally relevant microbial receptor on mast cells that plays a role in triggering inflammation.
Heritable germ and somatic cell lineage competitions in chimeric colonial protochordates
Authors: Stoner Douglas S. , Rinkevich Baruch , Weissman Irving L.
Published: July 2002

Theories of evolution that state natural selection acts on individuals have been modified to include multiple levels of selection. Here we demonstrate in chimeric protochordates that primitive germ cell (pgc) and somatic cell (psc) lineages have traits that also make them likely units of natural selection. Specifically, by using microsatellites to determine the genetic identity of various somatic and gametic tissues within vascularly fused
Botryllus schlosseri
chimeras, we show that genetically distinct pgc and psc can compete for access to developing gonads and somatic organs, and that this competition is hierarchical, reproducible, and heritable. Given that a single, highly polymorphic locus (Fu/HC) controls whether two contacting colonies fuse or reject, our findings also support a leading hypothesis for why the highly polymorphic histocompatibility loci common to many metazoa may have arisen or been maintained: to limit supercompetitor lineages to histocompatible kin.
Structure-based design of an RNA-binding zinc finger
Authors: McColl Damian J. , Honchell Cynthia D. , Frankel Alan D.
Published: July 2002

A structure-based approach was used to design RNA-binding zinc fingers that recognize the HIV-1 Rev response element (RRE). An arginine-rich α-helix from HIV-1 Rev was engineered into the zinc finger framework, and the designed fingers were shown to bind specifically to the RRE with high affinity and in a zinc-dependent manner, and display cobalt absorption and CD spectra characteristic of properly folded fingers. The results indicate that a monomeric zinc finger can recognize a specific nucleic acid site and that the α-helix of a finger can be used to recognize the major groove of RNA as well as DNA. The RRE-binding zinc fingers demonstrate how structure-based approaches may be used in the design of potential RNA-binding therapeutics and provide a framework for selecting RNA-binding fingers with desired specifications.