bluuebunny/crossref_metadata_2025 huggingface dataset:
A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.
Authors: Grafi G , Burnett R J , Helentjaris T , Larkins B A , DeCaprio J A , Sellers W R , Kaelin W G
Published: July 2002

Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in vivo with the replication-associated protein, RepA, encoded by the wheat dwarf virus. The maize Rb-related protein undergoes changes in level and phosphorylation state concomitant with endoreduplication, and it is phosphorylated in vitro by an S-phase kinase from endoreduplicating endosperm cells. Together, our results suggest that ZmRb is a representative of the pocket protein family and may play a role in cell cycle progression. Moreover, certain plant monopartite geminiviruses may operate similarly to mammalian DNA viruses, by targeting and inactivating the retinoblastoma protein, which otherwise induces G1 arrest.
Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.
Authors: Topper J N , Cai J , Falb D , Gimbrone M A
Published: July 2002

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.
Mirror symmetry breaking at the molecular level.
Authors: Avetisov V , Goldanskii V
Published: July 2002

Reasoning from two basic principles of molecular physics, P invariance of electromagnetic interaction and the second law of thermodynamics, one would conclude that mirror symmetry retained in the world of chiral molecules. This inference is fully consistent with what is observed in inorganic nature. However, in the bioorganic world, the reverse is true. Mirror symmetry there is definitely broken. Is it possible to account for this phenomenon without going beyond conventional concepts of the kinetics of enantioselective processes? This study is an attempt to survey all existing hypotheses containing this phenomenon.
A novel RNA polymerase I-dependent RNase activity that shortens nascent transcripts from the 3′ end
Authors: Tschochner Herbert
Published: July 2002

A novel RNase activity was identified in a yeast RNA polymerase I
(pol I)
in vitro
transcription system. Transcript cleavage
occurred at the 3′ end and was dependent on the presence of ternary pol
I/DNA/RNA complexes and an additional protein factor not
identical to transcription factor IIS (TFIIS). Transcript cleavage was
observed both on arrested complexes at the linearized ends of the
transcribed DNA and on intrinsic blocks of the DNA template. Shortened
transcripts that remained associated within the ternary complexes were
capable of resuming RNA chain elongation. Possible functions of the
nuclease for transcript elongation or termination are discussed.
Symmetry and the energy landscapes of biomolecules
Authors: Wolynes Peter G.
Published: July 2002

The role of symmetry in the folding of proteins is discussed using
energy landscape theory. An analytical argument shows it is much easier
to find sequences with funneled energy landscape capable of fast
folding if the structure is symmetric. The analogy with phase
transitions of small clusters with magic numbers is discussed.
Assessment of normal and mutant human presenilin function in
Caenorhabditis elegans
Authors: Levitan Diane , Doyle Timothy G. , Brousseau Denise , Lee Michael K. , Thinakaran Gopal , Slunt Hilda H. , Sisodia Sangram S. , Greenwald Iva
Published: July 2002

We provide evidence that normal human presenilins can substitute
for
Caenorhabditis elegans
SEL-12
protein in functional assays
in vivo
. In addition, six
familial Alzheimer disease-linked mutant human presenilins were tested
and found to have reduced ability to rescue the
sel-12
mutant phenotype, suggesting that they have lower than normal
presenilin activity. A human presenilin 1 deletion variant that fails
to be proteolytically processed and a mutant SEL-12 protein that lacks
the C terminus display considerable activity in this assay, suggesting
that neither presenilin proteolysis nor the C terminus is absolutely
required for normal presenilin function. We also show that
sel-12
is expressed in most neural and nonneural cell
types in all developmental stages. The reduced activity of mutant
presenilins and as yet unknown gain-of-function properties may be a
contributing factor in the development of Alzheimer disease.
Hyperplane arrangements, interval orders, and trees.
Authors: Stanley R P
Published: July 2002

A hyperplane arrangement is a finite set of hyperplanes in a real affine space. An especially important arrangement is the braid arrangement, which is the set of all hyperplanes xi - xj = 1, 1 </= i < j </= n, in Rn. Some combinatorial properties of certain deformations of the braid arrangement are surveyed. In particular, there are unexpected connections with the theory of interval orders and with the enumeration of trees. For instance, the number of labeled interval orders that can be obtained from n intervals I1,..., In of generic lengths is counted. There is also discussed an arrangement due to N. Linial whose number of regions is the number of alternating (or intransitive) trees, as defined by Gelfand, Graev, and Postnikov [Gelfand, I. M., Graev, M. I., and Postnikov, A. (1995), preprint]. Finally, a refinement is given, related to counting labeled trees by number of inversions, of a result of Shi [Shi, J.-Y. (1986), Lecture Notes in Mathematics, no. 1179, Springer-Verlag] that a certain deformation of the braid arrangement has (n + 1)n-1 regions.
Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.
Authors: Gerondakis S , Strasser A , Metcalf D , Grigoriadis G , Scheerlinck J Y , Grumont R J
Published: July 2002

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.
Interleukin 2 (IL-2) and interleukin 7 (IL-7) reciprocally induce IL-7 and IL-2 receptors on gamma delta T-cell receptor-positive intraepithelial lymphocytes.
Authors: Fujihashi K , Kawabata S , Hiroi T , Yamamoto M , McGhee J R , Nishikawa S , Kiyono H
Published: July 2002

In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of gamma delta T-cell receptor-positive T cells (gamma delta T cells). gamma delta T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into gamma delta (Dim) and gamma delta (Bright) fractions according to the intensity of gamma delta T-cell receptor expression. The gamma delta T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas gamma delta (Bright) T cells did not express either receptor. Our study also revealed that recombinant marine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on gamma delta (Dim) T cells but not on gamma delta (Bright) cells. Thus, treatment of gamma delta (Dim) T cells with rmIL-2 and rmIL-7 resulted in high proliferative responses, whereas gamma delta (Bright) T cells did not respond to these two cytokines. The sources of these two cytokines for gamma delta T cells were neighboring epithelial cells (IL-7) and alpha beta T cells (IL-2 and IL-7). Cytokine signaling by IL-2 and IL-7 from alpha beta T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of gamma delta T cells (e.g., gamma delta (Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division.
Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein
Authors: Cameron Craig E. , Ghosh Madhumita , Le Grice Stuart F. J. , Benkovic Stephen J.
Published: July 2002

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995)
Proc. Natl. Acad. Sci. USA
92, 1222–1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These “selectively deleted” heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT–DNA complex was apparent. The largest effect was observed for p66/p51Δ13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5′
32
P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from p51 had little effect on synthesis-dependent and synthesis-independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8–10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17–18 bp to 26–27 bp. The Δ13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis.
Natural killer cell acceptance of H-2 mismatch bone marrow grafts in transgenic mice expressing HLA-Cw3 specific killer cell inhibitory receptor (CD158b)
Authors: Cambiaggi Anna , Verthuy Christophe , Naquet Philippe , Romagné François , Ferrier Pierre , Biassoni Roberto , Moretta Alessandro , Moretta Lorenzo , Vivier Eric
Published: July 2002

Natural killer (NK) cells express killer cell inhibitory receptors (KIRs) for major histocompatibility complex class I molecules. Engagement of these surface receptors inhibits NK cell cytotoxic programs. KIR can also be expressed on T cell subsets, and their engagement similarly results in inhibition of effector functions initiated by the CD3/T cell receptor complex. KIR genes belong to two distinct families: the immunoglobulin superfamily (IgSF KIRs) and dimeric C2 lectins (lectin-like KIRs). Whereas both IgSF (p58: CD158, p70, and p140) and lectin-like KIRs (CD94/NKG2A heterodimers) have been found in human, only lectin-like KIRs (all members of the Ly-49 family) have been described in the mouse. We have generated transgenic mice expressing an IgSF KIR, CD158b (p58.2), which recognizes HLA-Cw3. Our data show that CD158b is necessary and sufficient to confer specificity to NK cells, as well as to modulate T cell activation programsin vitro. In addition, we did not detect any adaptation of CD158b cell surface expression to that of HLA class I ligands in the CD158b × HLA-Cw3 double transgenic mice, in contrast to observations with Ly-49 in the mouse. Therefore, distinct strategies of selection/calibration appear to be used by IgSF and lectin-like KIRs. Finally, the transgenic expression of CD158b KIR prevents thein vivorejection of H-2 mismatch bone marrow grafts, which express the cognate major histocompatibility class I HLA-Cw3 allele, demonstrating for the first time thein vivoimplication of human IgSF KIRs in the negative regulation of NK cell function.
Purification and characterization of acetone carboxylase from
Xanthobacter
strain Py2
Authors: Sluis Miriam K. , Ensign Scott A.
Published: July 2002

Acetone metabolism in the aerobic bacterium
Xanthobacter
strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an α
2
β
2
γ
2
quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels (≈10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO
2
, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a
V
max
for acetone carboxylation of 0.225 μmol acetoacetate formed min
−1
⋅mg
−1
at 30°C and pH 7.6 and apparent
K
m
values of 7.80 μM (acetone), 122 μM (ATP), and 4.17 mM (CO
2
plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.
Structural kinetics of transcription activation at the
malT
promoter of
Escherichia coli
by UV laser footprinting
Authors: Eichenberger Patrick , Déthiollaz Sylvie , Buc Henri , Geiselmann Johannes
Published: July 2002

We have studied the kinetics of transcriptional initiation and activation at the
malT
and
malTp1
promoters of
Escherichia coli
using UV laser footprinting. Contrary to previous studies and because of the very rapid signal acquisition by this technique, we can obtain structural information about true reaction intermediates of transcription initiation. The consequences of adding a transcriptional activator, the cAMP receptor protein/cAMP complex (CRP), are monitored in real time, permitting us to assign specific interactions to the activation of discrete steps in transcription initiation. Direct protein–protein contacts between CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the −10 hexamer. CRP slightly increased the rate of this isomerization reaction but, more importantly, favored the establishment of additional contacts between the DNA upstream of the CRP binding site and RNA polymerase subsequent to open complex formation. These contacts make a major contribution to transcriptional activation by stabilizing open forms of the promoter complex, thereby indirectly accelerating promoter escape. The ensemble of the kinetic, structural signals demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the
malT
promoter.
Vascular
MAD
s: Two novel
MAD
-related genes selectively inducible by flow in human vascular endothelium
Authors: Topper James N. , Cai Jiexing , Qiu Yubin , Anderson Keith R. , Xu Yong-Yao , Deeds James D. , Feeley Roslyn , Gimeno Carlos J. , Woolf Elizabeth A. , Tayber Olga , Mays Gail G. , Sampson Barbara A. , Schoen Frederick J. , Gimbrone Michael A. , Falb Dean
Published: July 2002

Vascular endothelium is an important transducer and integrator of both humoral and biomechanical stimuli within the cardiovascular system. Utilizing a differential display approach, we have identified two genes,
Smad6
and
Smad7
, encoding members of the MAD-related family of molecules, selectively induced in cultured human vascular endothelial cells by steady laminar shear stress, a physiologic fluid mechanical stimulus. MAD-related proteins are a recently identified family of intracellular proteins that are thought to be essential components in the signaling pathways of the serine/threonine kinase receptors of the transforming growth factor β superfamily. Smad6 and Smad7 possess unique structural features (compared with previously described MADs), and they can physically interact with each other, and, in the case of Smad6, with other known human MAD species, in endothelial cells. Transient expression of Smad6 or Smad7 in vascular endothelial cells inhibits the activation of a transfected reporter gene in response to both TGF-β and fluid mechanical stimulation. Both Smad6 and Smad7 exhibit a selective pattern of expression in human vascular endothelium
in vivo
as detected by immunohistochemistry and
in situ
hybridization. Thus, Smad6 and Smad7 constitute a novel class of MAD-related proteins, termed vascular MADs, that are induced by fluid mechanical forces and can modulate gene expression in response to both humoral and biomechanical stimulation in vascular endothelium.
The yeast peptide-methionine sulfoxide reductase functions as an antioxidant
in vivo
Authors: Moskovitz Jackob , Berlett Barbara S. , Poston J. Michael , Stadtman Earl R.
Published: July 2002

A gene homologous to methionine sulfoxide reductase (
msrA
) was identified as the predicted ORF (cosmid 9379) in chromosome V of
Saccharomyces cerevisiae
encoding a protein of 184 amino acids. The corresponding protein has been expressed in
Escherichia coli
and purified to homogeneity. The recombinant yeast MsrA possessed the same substrate specificity as the other known MsrA enzymes from mammalian and bacterial cells. Interruption of the yeast gene resulted in a null mutant, Δ
msrA::URA3
strain, which totally lost its cellular MsrA activity and was shown to be more sensitive to oxidative stress in comparison to its wild-type parent strain. Furthermore, high levels of free and protein-bound methionine sulfoxide were detected in extracts of
msrA
mutant cells relative to their wild-type parent cells, under various oxidative stresses. These findings show that MsrA is responsible for the reduction of methionine sulfoxide
in vivo
as well as
in vitro
in eukaryotic cells. Also, the results support the proposition that MsrA possess an antioxidant function. The ability of MsrA to repair oxidative damage
in vivo
may be of singular importance if methionine residues serve as antioxidants.
Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence
Authors: Ahlers Jeffrey D. , Takeshita Toshiyuki , Pendleton C. David , Berzofsky Jay A.
Published: July 2002

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.
Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression
Authors: Pützer Brigitte M. , Hitt Mary , Muller William J. , Emtage Peter , Gauldie Jack , Graham Frank L.
Published: July 2002

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host–tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of directin vivogene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12–B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 × 107pfu/mouse) of AdIL12–B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12–B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach forin vivocancer therapy.
Posttranslational regulation of cyclin D1 by retinoic acid: A chemoprevention mechanism
Authors: Langenfeld John , Kiyokawa Hiroaki , Sekula David , Boyle Jay , Dmitrovsky Ethan
Published: July 2002

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-
trans
retinoic acid (RA) signaling growth inhibition at G
1
in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G
1
-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and
in vitro
-translated cyclin D1 were performed.
In vitro
-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated
vs.
untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G
1
active in the prevention of human bronchial epithelial cell transformation.
Regulation of dopaminergic pathways by retinoids: Activation of the D2 receptor promoter by members of the retinoic acid receptor–retinoid X receptor family
Authors: Samad Tarek A. , Krezel Wojciech , Chambon Pierre , Borrelli Emiliana
Published: July 2002

Dopamine is a neuromodulator involved in the control of key physiological functions. Dopamine-dependent signal transduction is activated through the interaction with membrane receptors of the seven-transmembrane domain G protein-coupled family. Among them, dopamine D2 receptor is highly expressed in the striatum and the pituitary gland as well as by mesencephalic dopaminergic neurons. Lack of D2 receptors in mice leads to a locomotor parkinsonian-like phenotype and to pituitary tumors. The D2 receptor promoter has characteristics of a housekeeping gene. However, the restricted expression of this gene to particular neurons and cells points to a strict regulation of its expression by cell-specific transcription factors. We demonstrate here that the D2 receptor promoter contains a functional retinoic acid response element. Furthermore, analysis of retinoic acid receptor-null mice supports our finding and shows that in these animals D2 receptor expression is reduced. This finding assigns to retinoids an important role in the control of gene expression in the central nervous system.
Unexpected genetic and structural relationships of a long-forgotten flavoenzyme to NAD(P)H:quinone reductase (DT-diaphorase)
Authors: Zhao Qinjian , Yang Xiao Ling , Holtzclaw W. David , Talalay Paul
Published: July 2002

A mammalian cytosolic FAD-dependent enzyme that catalyzes the reduction of quinones by
N
-ribosyl- and
N
-alkyldihydronicotinamides, but not by NADH, NADPH, or NMNH (reduced nicotinamide mononucleotide), was isolated from bovine kidney more than 30 years ago [S. Liao, J. T. Dulaney and H. G. Williams-Ashman (1962)
J. Biol. Chem.
237, 2981–2987]. This enzyme is designated here as quinone reductase type 2 (QR
2
). Bovine QR
2
is a homodimer that migrates on SDS/PAGE at ≈22 kDa. Three tryptic peptides of bovine QR
2
(representing 39 amino acids) showed 43% identity to human NAD(P)H:quinone reductase (DT-diaphorase; EC
1.6.99.2
), here designated QR
1
and 82% identity to a related human cDNA clone [called hNQO
2
by A. K. Jaiswal, P. Burnett, M. Adesnik and O. W. McBride (1990)
Biochemistry
29, 1899–1906], and designated here as hQR
2
. The protein encoded by the latter cDNA did not show QR activity when tested with conventional nicotinamide nucleotides. The unexpected high homology between the old flavoenzyme and hQR
2
prompted us to clone and overexpress hQR
2
. The properties of hQR
2
were identical to those of the flavoenzyme described by S. Liao and H. G. Williams-Ashman, thus establishing their genetic identity. Recombinant human QR
2
: (
i
) reacts with
N
-ribosyl- and
N
-alkyldihydronicotinamides, but not with NADH, NADPH, or NMNH; (
ii
) is very weakly inhibited by dicumarol or Cibacron blue; (
iii
) is very potently inhibited by benzo[
a
]pyrene. The x-ray crystal structure of rat QR
1
shows that the 43 amino acid C-terminal tail of QR
1
provides the binding site for the hydrophilic portions of NADH and NADPH. In the absence of this binding site in QR
2
, the enzyme retains the essential catalytic machinery, including affinity for FAD, but cannot bind phosphorylated hydride donors.
Evidence for a lipochaperonin: Association of active proteinfolding GroESL oligomers with lipids can stabilize membranes under heat shock conditions
Authors: Török Zsolt , Horváth Ibolya , Goloubinoff Pierre , Kovács Eszter , Glatz Attila , Balogh Gábor , Vígh László
Published: July 2002

During heat shock, structural changes in proteins and membranes may lead to cell death. While GroE and other chaperone proteins are involved in the prevention of stress-induced protein aggregation and in the recovery of protein structures, a mechanism for short-term membrane stabilization during stress remains to be established. We found that GroEL chaperonin can associate with model lipid membranes. Binding was apparently governed by the composition and the physical state of the host bilayer. Limited proteolysis of GroEL oligomers by proteinase K, which removes selectively the conserved glycine- and methionine-rich C terminus, leaving the chaperonin oligomer intact, prevented chaperonin association with lipid membranes. GroEL increased the lipid order in the liquid crystalline state, yet remained functional as a protein-folding chaperonin. This suggests that, during stress, chaperonins can assume the functions of assisting the folding of both soluble and membrane-associated proteins while concomitantly stabilizing lipid membranes.
Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice
Authors: Araki Ryoko , Fujimori Akira , Hamatani Kiyohiro , Mita Kazuei , Saito Toshiyuki , Mori Masahiko , Fukumura Ryutaro , Morimyo Mitsuoki , Muto Masahiro , Itoh Masahiro , Tatsumi Kouichi , Abe Masumi
Published: July 2002

The severe combined immune deficiency (SCID) mouse was reported as an animal model for human immune deficiency. Through the course of several studies, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene came to be considered a candidate for the SCID-responsible gene. We isolated an ORF of the murine DNA-PKcs gene from SCID mice and their parent strain C.B-17 mice and determined the DNA sequences. The ORF of the murine DNA-PKcs gene contained 4128-aa residues and had 78.9% homology with the human DNA-PKcs gene. A particularly important finding is that a T to A transversion results in the substitution of termination codon in SCID mice for the Tyr-4046 in C.B-17 mice. No other mutation was detected in the ORF of the gene. The generality of this transversion was confirmed using four individual SCID and wild-type mice. The substitution took place in the phosphatidylinositol 3-kinase domain, and the mutated gene encodes the truncated products missing 83 residues of wild-type DNA-PKcs products. Furthermore, the quantity of DNA-PKcs transcript in wild-type and SCID cells was almost equal. These observations indicate that the DNA-PKcs gene is the SCID-responsible gene itself and that the detected mutation leads to the SCID aberration.
Dietary fat influences on polyp phenotype in multiple intestinal neoplasia mice
Authors: Wasan Harpreet S. , Novelli Marco , Bee Julie , Bodmer Walter F.
Published: July 2002

Significant differences in colon cancer incidence worldwide have led to the hypothesis that this variation can be explained largely by environmental, notably dietary influences. Although a positive correlation between dietary fat intake and incidence is suggested from some human epidemiological and rodent carcinogenesis studies, a direct association remains contentious. Using a spontaneous mouse tumor model of multiple intestinal neoplasia, we demonstrate that there is a generalized increase in tumor counts, in both the large and small bowel with higher dietary fat [standard (3%) fat versus high (15%) fat diet (mean ± SD) 1.59 ± 1.46 vs. 3.85 ± 2.37
P
< 0.001 and 21.36 ± 7.4 vs. 31.3 ± 9.7, respectively,
P
< 0.001]. Increasing dietary fat also increases polyp size in the small bowel. These changes appear independent of total calorific intake as assessed by body weights. Halving the crude fiber intake together with an increase in dietary fat from 3% to 10% did not have as marked an effect on tumor counts as an increase of fat alone to 15%, which also decreased survival (
P
< 0.05). These results demonstrate that increasing dietary fat intake from weaning can have a significant adverse effect on polyp numbers in mice genetically predisposed to intestinal tumor development. A further understanding of the biology of this interaction may provide novel strategies aimed at both colonic polyp prevention and treatment.
Changing the mechanism of transcriptional activation by phage λ repressor
Authors: Li Mei , McClure W. R. , Susskind Miriam M.
Published: July 2002

The first steps of transcription initiation include binding of RNA polymerase to a promoter to form an inactive, unstable, closed complex (described by an equilibrium constant,
K
B
) and isomerization of the closed complex to an active, stable, open complex (described by a forward rate constant,
k
f
). λ cI protein activates the
P
RM
promoter by specifically increasing
k
f
. A positive control mutant, cI-pc2, is defective for activation because it fails to raise
k
f
. An Arg to His change in the σ
70
subunit of RNA polymerase was previously obtained as an allele-specific suppressor of
cI-pc2
. To elucidate how the mutant polymerase restores the activation function of the mutant activator, abortive initiation assays were performed, using purified cI proteins and RNA polymerase holoenzymes. The change in σ does not significantly alter
K
B
or
k
f
in the absence of cI protein. As expected, cI-pc2 activates the mutant polymerase in the same way that wild-type cI activates the wild-type polymerase, by increasing
k
f
. An unexpected and novel finding is that the wild-type activator stimulates the mutant polymerase, but not wild-type polymerase, by increasing
K
B
.
Control of direction of flagellar rotation in bacterial chemotaxis
Authors: Scharf Birgit E. , Fahrner Karen A. , Turner Linda , Berg Howard C.
Published: July 2002

The motile behavior of the bacterium
Escherichia coli
depends on the direction of rotation of its flagellar motors. Binding of the phosphorylated signaling molecule CheY to a motor component FliM is known to enhance clockwise rotation. It is difficult to study this interaction
in vivo
, because the dynamics of phosphorylation of CheY by its kinase CheA and the hydrolysis of CheY (accelerated by CheZ) are not under direct experimental control. Here, we examine instead the interaction with the flagellar motor of a double mutant CheY
13DK106YW
that is active without phosphorylation. The behavioral assays were carried out on tethered cells lacking CheA and CheZ. The effects of variation in intracellular concentration of the mutant protein were highly nonlinear. However, they can be explained by a thermal isomerization model in which the free energies of clockwise and counterclockwise states depend linearly on the amount of CheY bound.
Melanopsin: An opsin in melanophores, brain, and eye
Authors: Provencio Ignacio , Jiang Guisen , De Grip Willem J. , Hayes William Pär , Rollag Mark D.
Published: July 2002

We have identified an opsin, melanopsin, in photosensitive dermal melanophores of
Xenopus laevis
. Its deduced amino acid sequence shares greatest homology with cephalopod opsins. The predicted secondary structure of melanopsin indicates the presence of a long cytoplasmic tail with multiple putative phosphorylation sites, suggesting that this opsin’s function may be finely regulated. Melanopsin mRNA is expressed in hypothalamic sites thought to contain deep brain photoreceptors and in the iris, a structure known to be directly photosensitive in amphibians. Melanopsin message is also localized in retinal cells residing in the outermost lamina of the inner nuclear layer where horizontal cells are typically found. Its expression in retinal and nonretinal tissues suggests a role in vision and nonvisual photoreceptive tasks, such as photic control of skin pigmentation, pupillary aperture, and circadian and photoperiodic physiology.
CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth
Authors: Babic Alexander M. , Kireeva Maria L. , Kolesnikova Tatiana V. , Lau Lester F.
Published: July 2002

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested expressCYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of theCYR61cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.
Cell-specific viral targeting mediated by a soluble retroviral receptor-ligand fusion protein
Authors: Snitkovsky Sophie , Young John A. T.
Published: July 2002

TVA, the cellular receptor for subgroup A avian leukosis viruses (ALV-A) can mediate viral entry when expressed as a transmembrane protein or as a glycosylphosphatidylinositol-linked protein on the surfaces of transfected mammalian cells. To determine whether mammalian cells can be rendered susceptible to ALV-A infection by attaching a soluble form of TVA to their plasma membranes, the TVA-epidermal growth factor (EGF) fusion protein was generated. TVA-EGF is comprised of the extracellular domain of TVA linked to the mature form of human EGF. Flow cytometric analysis confirmed that TVA-EGF is a bifunctional reagent capable of binding simultaneously to cell surface EGF receptors and to an ALV-A surface envelope-Ig fusion protein. TVA-EGF prebound to transfected mouse fibroblasts expressing either wild-type or kinase-deficient human EGF receptors, rendered these cells highly susceptible to infection by ALV-A vectors. Viral infection was blocked specifically in the presence of a recombinant human EGF protein, demonstrating that the binding of TVA-EGF to EGF receptors was essential for infectivity. These studies have demonstrated that a soluble TVA-ligand fusion protein can mediate viral infection when attached to specific cell surfaces, suggesting an approach for targeting retroviral infection to specific cell types.
High temperature promotes auxin-mediated hypocotyl elongation in
Arabidopsis
Authors: Gray William M. , Östin Anders , Sandberg Göran , Romano Charles P. , Estelle Mark
Published: July 2002

Physiological studies with excised stem segments have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of cell elongation. Supporting evidence from intact plants has been somewhat more difficult to obtain, however. Here, we report the identification and characterization of an auxin-mediated cell elongation growth response in
Arabidopsis thaliana
. When grown in the light at high temperature (29°C),
Arabidopsis
seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20°C. This temperature-dependent growth response is sharply reduced by mutations in the auxin response or transport pathways and in seedlings containing reduced levels of free IAA. In contrast, mutants deficient in gibberellin and abscisic acid biosynthesis or in ethylene response are unaffected. Furthermore, we detect a corresponding increase in the level of free IAA in seedlings grown at high temperature, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response. Consistent with this possibility, high temperature also stimulates other auxin-mediated processes including auxin-inducible gene expression. Based on these results, we propose that growth at high temperature promotes an increase in auxin levels resulting in increased hypocotyl elongation. These results strongly support the contention that endogenous auxin promotes cell elongation in intact plants.
Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control
Authors: Wright Jocyndra A. , Keegan Kathleen S. , Herendeen Daniel R. , Bentley Nicola J. , Carr Antony M. , Hoekstra Merl F. , Concannon Patrick
Published: July 2002

In fission yeast, the
rad3
gene product plays a critical role in sensing DNA structure defects and activating damage response pathways. A structural homologue of
rad3
in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex. Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia–telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.
KARP-1 is induced by DNA damage in a p53- and ataxia telangiectasia mutated-dependent fashion
Authors: Myung Kyungjae , Braastad Corey , He Dong Ming , Hendrickson Eric A.
Published: July 2002

The KARP-1 (
K
u86
A
utoantigen
R
elated
P
rotein-
1
) gene, which is expressed from the human Ku86 autoantigen locus, appears to play a role in mammalian DNA double-strand break repair as a regulator of the DNA-dependent protein kinase complex. Here we demonstrate that KARP-1 gene expression is significantly up-regulated following exposure of cells to DNA damage. KARP-1 mRNA induction was completely dependent on the ataxia telangiectasia and p53 gene products, consistent with the presence of a p53 binding site within the second intron of the KARP-1 locus. These observations link ataxia telangiectasia, p53, and KARP-1 in a common pathway.
The Notch1 receptor is cleaved constitutively by a furin-like convertase
Authors: Logeat Frédérique , Bessia Christine , Brou Christel , LeBail Odile , Jarriault Sophie , Seidah Nabil G. , Israël Alain
Published: July 2002

The Notch receptor, which is involved in numerous cell fate decisions in invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). We show here that proteolytic processing of p300 is an essential step in the formation of the biologically active receptor because only the cleaved fragments are present at the cell surface. Our results confirm and extend recent reports indicating that the Notch receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. We report here that constitutive processing of murine Notch1 involves a furin-like convertase. We show that the calcium ionophore A23187 and the α1-antitrypsin variant, α 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed.
In vitro
digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651–1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolished processing of the Notch1 receptor.
Evidence for multiclonality in multicentric Kaposi’s sarcoma
Authors: Gill Parkash S. , Tsai Yvonne C. , Rao Adupa P. , Spruck Charles H. , Zheng Tong , Harrington William A. , Cheung Tony , Nathwani Bharat , Jones Peter A.
Published: July 2002

Kaposi’s sarcoma (KS) develops in a variety of clinical states and is the most common tumor seen in patients with HIV-1 infection. KS develops as a multifocal mucocutaneous disease with subsequent spread to visceral organs, and it has been argued to be a benign proliferation caused by its multifocality at initial presentation, lack of aneuploidy, and spontaneous regression upon withdrawal of immunosuppressive agents in iatrogenically induced disease. We wished to determine whether KS lesions are clonal, indicative of a true neoplasm. Also, we tested whether multifocal KS lesions are clonally related, derived from a common progenitor cell or of independent cellular origin. We studied the X-chromosome inactivation pattern of the human androgen receptor gene in tumor biopsies of women with KS. This procedure tests for the clonality of a tissue specimen, a hallmark of neoplasia. Each specimen was microdissected to minimize normal cell contamination. Of 12 evaluable cases, 10 were HIV-seropositive and 2 were HIV-seronegative. Twenty-four biopsies from the 12 patients were examined. Five cases were consistent with individual KS lesions being clonal. In two cases, multiple KS specimens derived from the individual patients had different androgen receptor alleles inactivated, proving unequivocally that these KS lesions arose independently from distinct transformed cells. In seven cases, only a polyclonal pattern of inactivation was observed, whereas two others had tumor areas of both clonal and polyclonal inactivation patterns. These findings suggest that KS can be a clonal neoplasm, and in some of the cases multiple KS lesions in a given patient can arise from independent cellular origins and acquire clonal characteristics. The polyclonal inactivation pattern observed in other KS lesions may represent a premalignant stage or false negative results.