bluuebunny/crossref_metadata_2025 huggingface dataset:
Shearing rate of time-dependent E×B flow
Authors: Hahm T. S. , Beer M. A. , Lin Z. , Hammett G. W. , Lee W. W. , Tang W. M.
Published: July 2002

Theory of E×B shear suppression of turbulence in toroidal geometry [Phys. Plasmas 2, 1648 (1995)] is extended to include fast time variations of the E×B flows often observed in nonlinear simulations of tokamak turbulence. It is shown that the quickly time varying components of the E×B flows, while they typically contribute significantly to the instantaneous E×B shearing rate, are less effective than the slowly time varying components in suppressing turbulence. This is because the shear flow pattern changes before eddies get distorted enough. The effective E×B shearing rate capturing this important physics is analytically derived and estimated from zonal flow statistics of gyrofluid simulation. This provides new insights into understanding recent gyrofluid and gyrokinetic simulations that yield a reduced, but not completely quenched, level of turbulence in the presence of turbulence-driven zonal flows.
Stability of magnetohydrodynamic Dean Flow as applied to centrifugally confined plasmas
Authors: Hassam A. B.
Published: July 2002

Dean Flow is the azimuthal flow of fluid between static concentric cylinders. In a magnetized plasma, there may also be radial stratification of the pressure. The ideal magnetohydrodynamic stability of such a flow in the presence of a strong axial magnetic field and an added radial gravitational force is examined. It is shown that both the Kelvin–Helmholtz instability and pressure-gradient-driven interchanges can be stabilized if the flow is driven by a unidirectional external force and if the plasma annulus is sufficiently thin (large aspect ratio). These results find application in schemes using centrifugal confinement of plasma for fusion.
Inboard/outboard asymmetry of poloidal flow observed in the Compact Helical System
Authors: Nishimura S. , Ida K. , Osakabe M. , Minami T. , Tanaka K. ,   CHS Group
Published: July 2002

The poloidal flow velocity and the density of fully ionized carbon were measured in the Compact Helical System [K. Ida et al., Phys. Rev. Lett. 67, 58 (1991)] using the charge exchange spectroscopy with the bidirectional viewing. The poloidal asymmetry of the poloidal flow velocity observed in the outward shifted plasma is due to the electrostatic potential being constant on the magnetic flux surfaces. The poloidal asymmetry of the ion density can be explained by the conservation of the poloidal ion flux under the strong toroidal viscosity.
The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes
Authors: Zhou Z. Hong , McCarthy Diane B. , O'Connor Catherine M. , Reed Lester J. , Stoops James K.
Published: July 2002

The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (Mr≈ 7.8 × 106) comprising about 22 molecules of pyruvate dehydrogenase (E1) and about 6 molecules of dihydrolipoamide dehydrogenase (E3) with its binding protein associated with the 60-subunit dihydrolipoamide acetyltransferase (E2) core provides considerable insight into the structural and functional organization of the largest multienzyme complex known. The structure shows that potentially 60 centers for acetyl-CoA synthesis are organized in sets of three at each of the 20 vertices of the pentagonal dodecahedral core. These centers consist of three E1molecules bound to one E2trimer adjacent to an E3molecule in each of 12 pentagonal openings. The E1components are anchored to the E1-binding domain of the E2subunits through an ≈50-Å-long linker. Three of these linkers emanate from the outside edges of the triangular base of the E2trimer and form a cage around its base that may shelter the lipoyl domains and the E1and E2active sites. The docking of the atomic structures of E1and the E1binding and lipoyl domains of E2in the electron microscopy map gives a good fit and indicates that the E1active site is ≈95 Å above the base of the trimer. We propose that the lipoyl domains and its tether (swinging arm) rotate about the E1-binding domain of E2,which is centrally located 45–50 Å from the E1, E2, and E3active sites, and that the highly flexible breathing core augments the transfer of intermediates between active sites.
Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors
Authors: Parker Jacqueline N. , Gillespie G. Yancey , Love Cammy E. , Randall Suzanne , Whitley Richard J. , Markert James M.
Published: July 2002

Genetically engineered, neuroattenuated herpes simplex viruses (HSVs) expressing various cytokines can improve survival when used in the treatment of experimental brain tumors. These attenuated viruses have both copies of γ134.5 deleted. Recently, we demonstrated increased survival of C57BL/6 mice bearing syngeneic GL-261 gliomas when treated with an engineered HSV expressing IL-4, as compared with treatment with the parent construct (γ134.5−) alone or with a virus expressing IL-10. Herein, we report construction of a conditionally replication-competent mutant expressing both subunits of mIL-12 (M002) and its evaluation in a syngeneic neuroblastoma murine model. IL-12 induces a helper T cell subset type 1 response, which may induce more durable antitumor effects.In vitrostudies showed that, when infected with M002, both Vero cells and murine Neuro-2a neuroblastoma cells produced physiologically relevant levels of IL-12 heterodimers, as determined by ELISA. M002 was cytotoxic for Neuro-2a cells and human glioma cell lines U251MG and D54MG. Neurotoxicity studies, as defined by plaque-forming units/LD50, performed in HSV-1-sensitive A/J strain mice found that M002 was not toxic even at high doses. When evaluated in an intracranial syngeneic neuroblastoma murine model, median survival of M002-treated animals was significantly longer than the median survival of animals treated with R3659, the parent γ134.5−mutant lacking any cytokine gene insert. Immunohistochemical analysis of M002-treated tumors identified a pronounced influx of CD4+T cells and macrophages as well as CD8+cells when compared with an analysis of R3659-treated tumors. We conclude that M002 produced a survival benefit via oncolytic effects combined with immunologic effects meditated by helper T cells of subset type 1.
Comparative genomic analysis of
Vibrio cholerae
: Genes that correlate with cholera endemic and pandemic disease
Authors: Dziejman Michelle , Balon Emmy , Boyd Dana , Fraser Clare M. , Heidelberg John F. , Mekalanos John J.
Published: July 2002

Historically, the first six recorded cholera pandemics occurred between 1817 and 1923 and were caused by
Vibrio cholerae
O1 serogroup strains of the classical biotype. Although strains of the El Tor biotype caused sporadic infections and cholera epidemics as early as 1910, it was not until 1961 that this biotype emerged to cause the 7th pandemic, eventually resulting in the global elimination of classical biotype strains as a cause of disease. The completed genome sequence of 7th pandemic El Tor O1 strain N16961 has provided an important tool to begin addressing questions about the evolution of
V. cholerae
as a human pathogen and environmental organism. To facilitate such studies, we constructed a
V. cholerae
genomic microarray that displays over 93% of the predicted genes of strain N16961 as spotted features. Hybridization of labeled genomic DNA from different strains to this microarray allowed us to compare the gene content of N16961 to that of other
V. cholerae
isolates. Surprisingly, the results reveal a high degree of conservation among the strains tested. However, genes unique to all pandemic strains as well as genes specific to 7th pandemic El Tor and related O139 serogroup strains were identified. These latter genes may encode gain-of-function traits specifically associated with displacement of the preexisting classical strains in South Asia and may also promote the establishment of endemic disease in previously cholera-free locations.
Discovery of five conserved β-defensin gene clusters using a computational search strategy
Authors: Schutte Brian C. , Mitros Joseph P. , Bartlett Jennifer A. , Walters Jesse D. , Jia Hong Peng , Welsh Michael J. , Casavant Thomas L. , McCray Paul B.
Published: July 2002

The innate immune system includes antimicrobial peptides that protect multicellular organisms from a diverse spectrum of microorganisms. β-Defensins comprise one important family of mammalian antimicrobial peptides. The annotation of the human genome fails to reveal the expected diversity, and a recent query of the draft sequence with theblastsearch engine found only one new β-defensin gene (DEFB3). To define better the β-defensin gene family, we adopted a genomics approach that useshmmer, a computational search tool based on hidden Markov models, in combination withblast. This strategy identified 28 new human and 43 new mouse β-defensin genes in five syntenic chromosomal regions. Within each syntenic cluster, the gene sequences and organization were similar, suggesting each cluster pair arose from a common ancestor and was retained because of conserved functions. Preliminary analysis indicates that at least 26 of the predicted genes are transcribed. These results demonstrate the value of a genomewide search strategy to identify genes with conserved structural motifs. Discovery of these genes represents a new starting point for exploring the role of β-defensins in innate immunity.
Genetic variation at the 22q11PRODH2/DGCR6locus presents an unusual pattern and increases susceptibility to schizophrenia
Authors: Liu Hui , Heath Simon C. , Sobin Christina , Roos J. Louw , Galke Brandi L. , Blundell Maude L. , Lenane Marge , Robertson Brian , Wijsman Ellen M. , Rapoport Judith L. , Gogos Joseph A. , Karayiorgou Maria
Published: July 2002

The location of a schizophrenia susceptibility locus at chromosome 22q11 has been suggested by genome-wide linkage studies. Additional support was provided by the observation of a higher-than-expected frequency of 22q11 microdeletions in patients with schizophrenia and the demonstration that ≈20–30% of individuals with 22q11 microdeletions develop schizophrenia or schizoaffective disorder in adolescence and adulthood. Analysis of the extent of these microdeletions by using polymorphic markers afforded further refinement of this locus to a region of ≈1.5 Mb. Recently, a high rate of 22q11 microdeletions was also reported for a cohort of 47 patients with Childhood Onset Schizophrenia, a rare and severe form of schizophrenia with onset by age 13. It is therefore likely that this 1.5-Mb region contains one or more genes that predispose to schizophrenia. In three independent samples, we provide evidence for a contribution of thePRODH2/DGCR6locus in 22q11-associated schizophrenia. We also uncover an unusual pattern ofPRODH2gene variation that mimics the sequence of a linked pseudogene. Several of the pseudogene-like variants we identified result in missense changes at conserved residues and may prevent synthesis of a fully functional enzyme. Our results have implications for understanding the genetic basis of the 22q11-associated psychiatric phenotypes and provide further insights into the genomic instability of this region.
Activation of Trk neurotrophin receptors in the absence of neurotrophins
Authors: Lee Francis S. , Chao Moses V.
Published: July 2002

Neurotrophins regulate neuronal cell survival and synaptic
plasticity through activation of Trk receptor tyrosine kinases. Binding
of neurotrophins to Trk receptors results in receptor
autophosphorylation and downstream phosphorylation cascades. Here, we
describe an approach to use small molecule agonists to transactivate
Trk neurotrophin receptors. Activation of TrkA receptors in PC12 cells
and TrkB in hippocampal neurons was observed after treatment with
adenosine, a neuromodulator that acts through G protein-coupled
receptors. These effects were reproduced by using the adenosine agonist
CGS 21680 and were counteracted with the antagonist ZM 241385,
indicating that this transactivation event by adenosine involves
adenosine 2A receptors. The increase in Trk activity could be inhibited
by the use of the Src family-specific inhibitor, PP1, or K252a, an
inhibitor of Trk receptors. In contrast to other G protein-coupled
receptor transactivation events, adenosine used Trk receptor signaling
with a longer time course. Moreover, adenosine activated
phosphatidylinositol 3-kinase/Akt through a Trk-dependent mechanism
that resulted in increased cell survival after nerve growth factor or
brain-derived neurotrophic factor withdrawal. Therefore, adenosine
acting through the A
2A
receptors exerts a trophic effect
through the engagement of Trk receptors. These results provide an
explanation for neuroprotective actions of adenosine through a
unique signaling mechanism and raise the possibility that small
molecules may be used to elicit neurotrophic effects for the treatment
of neurodegenerative diseases.
Complete genome sequence of
Caulobacter crescentus
Authors: Nierman William C. , Feldblyum Tamara V. , Laub Michael T. , Paulsen Ian T. , Nelson Karen E. , Eisen Jonathan , Heidelberg John F. , Alley M. R. K. , Ohta Noriko , Maddock Janine R. , Potocka Isabel , Nelson William C. , Newton Austin , Stephens Craig , Phadke Nikhil D. , Ely Bert , DeBoy Robert T. , Dodson Robert J. , Durkin A. Scott , Gwinn Michelle L. , Haft Daniel H. , Kolonay James F. , Smit John , Craven M. B. , Khouri Hoda , Shetty Jyoti , Berry Kristi , Utterback Teresa , Tran Kevin , Wolf Alex , Vamathevan Jessica , Ermolaeva Maria , White Owen , Salzberg Steven L. , Venter J. Craig , Shapiro Lucy , Fraser Claire M.
Published: July 2002

The complete genome sequence of
Caulobacter
crescentus
was determined to be 4,016,942 base pairs in a
single circular chromosome encoding 3,767 genes. This organism, which
grows in a dilute aquatic environment, coordinates the cell division
cycle and multiple cell differentiation events. With the annotated
genome sequence, a full description of the genetic network that
controls bacterial differentiation, cell growth, and cell cycle
progression is within reach. Two-component signal transduction proteins
are known to play a significant role in cell cycle progression. Genome
analysis revealed that the
C. crescentus
genome encodes
a significantly higher number of these signaling proteins (105) than
any bacterial genome sequenced thus far. Another regulatory mechanism
involved in cell cycle progression is DNA methylation. The occurrence
of the recognition sequence for an essential DNA methylating enzyme
that is required for cell cycle regulation is severely limited and
shows a bias to intergenic regions. The genome contains multiple
clusters of genes encoding proteins essential for survival in a
nutrient poor habitat. Included are those involved in chemotaxis, outer
membrane channel function, degradation of aromatic ring compounds, and
the breakdown of plant-derived carbon sources, in addition to many
extracytoplasmic function sigma factors, providing the organism with
the ability to respond to a wide range of environmental fluctuations.
C. crescentus
is, to our knowledge, the first
free-living α-class proteobacterium to be sequenced and will serve as
a foundation for exploring the biology of this group of bacteria, which
includes the obligate endosymbiont and human pathogen
Rickettsia
prowazekii
, the plant pathogen
Agrobacterium
tumefaciens
, and the bovine and human pathogen
Brucella
abortus
.
Hypoxia-inducible factor 1α protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations
Authors: Sutter Carrie Hayes , Laughner Erik , Semenza Gregg L.
Published: July 2002

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to reduced O
2
availability in mammals, including angiogenesis, erythropoiesis, and glycolysis. HIF-1 activity is controlled by the O
2
-regulated expression of the HIF-1α subunit. Under nonhypoxic conditions, HIF-1α protein is subject to ubiquitination and proteasomal degradation. Here we report that missense mutations and/or deletions involving several different regions of HIF-1α result in constitutive expression and transcriptional activity in nonhypoxic cells. We demonstrate that hypoxia results in decreased ubiquitination of HIF-1α and that missense mutations increase HIF-1α expression under nonhypoxic conditions by blocking ubiquitination.
A nonsense mutation in zebrafish
gata1
causes the bloodless phenotype in
vlad tepes
Authors: Lyons Susan E. , Lawson Nathan D. , Lei Lin , Bennett Paul E. , Weinstein Brant M. , Liu P. Paul
Published: July 2002

Vlad tepes
(

vlt
m651

) is one of only five “bloodless” zebrafish mutants isolated through large-scale chemical mutagenesis screening. It is characterized by a severe reduction in blood cell progenitors and few or no blood cells at the onset of circulation. We now report characterization of the mutant phenotype and the identification of the gene mutated in

vlt
m651

. Embryos homozygous for the

vlt
m651

mutation had normal expression of hematopoietic stem cell markers through 24 h postfertilization, as well as normal expression of myeloid and lymphoid markers. Analysis of erythroid development revealed variable expression of erythroid markers. Through positional and candidate gene cloning approaches we identified a nonsense mutation in the
gata1
gene, 1015C → T (Arg-339 → Stop), in

vlt
m651

. The nonsense mutation was located C-terminal to the two zinc fingers and resulted in a truncated protein that was unable to bind DNA or mediate GATA-specific transactivation. A BAC clone containing the zebrafish
gata1
gene was able to rescue the bloodless phenotype in

vlt
m651

. These results show that the

vlt
m651

mutation is a previously uncharacterized
gata1
allele in the zebrafish. The

vlt
m651

mutation sheds new light on Gata1 structure and function
in vivo
, demonstrates that Gata1 plays an essential role in zebrafish hematopoiesis with significant conservation of function between mammals and zebrafish, and offers a powerful tool for future studies of the hematopoietic pathway.
A neural correlate of syntactic encoding during speech production
Authors: Indefrey Peter , Brown Colin M. , Hellwig Frauke , Amunts Katrin , Herzog Hans , Seitz Rüdiger J. , Hagoort Peter
Published: July 2002

Spoken language is one of the most compact and structured ways to convey information. The linguistic ability to structure individual words into larger sentence units permits speakers to express a nearly unlimited range of meanings. This ability is rooted in speakers' knowledge of syntax and in the corresponding process of syntactic encoding. Syntactic encoding is highly automatized, operates largely outside of conscious awareness, and overlaps closely in time with several other processes of language production. With the use of positron emission tomography we investigated the cortical activations during spoken language production that are related to the syntactic encoding process. In the paradigm of restrictive scene description, utterances varying in complexity of syntactic encoding were elicited. Results provided evidence that the left Rolandic operculum, caudally adjacent to Broca's area, is involved in both sentence-level and local (phrase-level) syntactic encoding during speaking.
Structural insight into the complex formation of latent matrix metalloproteinase 2 with tissue inhibitor of metalloproteinase 2
Authors: Morgunova Ekaterina , Tuuttila Ari , Bergmann Ulrich , Tryggvason Karl
Published: July 2002

Matrix metalloproteinases (MMPs) are a family of multidomain enzymes involved in the physiological degradation of connective tissue, as well as in pathological states such as tumor invasion and arthritis. Apart from transcriptional regulation, MMPs are controlled by proenzyme activation and a class of specific tissue inhibitors of metalloproteinases (TIMPs) that bind to the catalytic site. TIMP-2 is a potent inhibitor of MMPs, but it has also been implicated in a unique cell surface activation mechanism of latent MMP-2/gelatinase A/type IV collagenase (proMMP-2), through its binding to the hemopexin domain of proMMP-2 on the one hand and to a membrane-type MMP activator on the other. The present crystal structure of the human proMMP-2/TIMP-2 complex reveals an interaction between the hemopexin domain of proMMP-2 and the C-terminal domain of TIMP-2, leaving the catalytic site of MMP-2 and the inhibitory site of TIMP-2 distant and spatially isolated. The interfacial contact of these two proteins is characterized by two distinct binding regions composed of alternating hydrophobic and hydrophilic interactions. This unique structure provides information for how specificity for noninhibitory MMP/TIMP complex formation is achieved.
Unarmed, tumor-specific monoclonal antibody effectively treats brain tumors
Authors: Sampson John H. , Crotty Laura E. , Lee Samson , Archer Gary E. , Ashley David M. , Wikstrand Carol J. , Hale Laura P. , Small Clayton , Dranoff Glenn , Friedman Allan H. , Friedman Henry S. , Bigner Darell D.
Published: July 2002

The epidermal growth factor receptor (EGFR) is often amplified and rearranged structurally in tumors of the brain, breast, lung, and ovary. The most common mutation, EGFRvIII, is characterized by an in-frame deletion of 801 base pairs, resulting in the generation of a novel tumor-specific epitope at the fusion junction. A murine homologue of the human EGFRvIII mutation was created, and an IgG2a murine mAb, Y10, was generated that recognizes the human and murine equivalents of this tumor-specific antigen.In vitro, Y10 was found to inhibit DNA synthesis and cellular proliferation and to induce autonomous, complement-mediated, and antibodydependent cell-mediated cytotoxicity. Systemic treatment with i.p. Y10 of s.c. B16 melanomas transfected to express stably the murine EGFRvIII led to long-term survival in all mice treated (n= 20;P< 0.001). Similar therapy with i.p. Y10 failed to increase median survival of mice with EGFRvIII-expressing B16 melanomas in the brain; however, treatment with a single intratumoral injection of Y10 increased median survival by an average 286%, with 26% long-term survivors (n= 117;P< 0.001). The mechanism of action of Y10in vivowas shown to be independent of complement, granulocytes, natural killer cells, and T lymphocytes throughin vivocomplement and cell subset depletions. Treatment with Y10 in Fc receptor knockout mice demonstrated the mechanism of Y10 to be Fc receptor-dependent. These data indicate that an unarmed, tumor-specific mAb may be an effective immunotherapy against human tumors and potentially other pathologic processes in the “immunologically privileged” central nervous system.
In vivourea cycle flux distinguishes and correlates with phenotypic severity in disorders of the urea cycle
Authors: Lee Brendan , Yu Hong , Jahoor Farook , O'Brien William , Beaudet Arthur L. , Reeds Peter
Published: July 2002

Urea cycle disorders are a group of inborn errors of hepatic metabolism that result in often life-threatening hyperammonemia and hyperglutaminemia. Clinical and laboratory diagnosis of partial deficiencies during asymptomatic periods is difficult, and correlation of phenotypic severity with either genotype and/orin vitroenzyme activity is often imprecise. We hypothesized that stable isotopically determinedin vivorates of total body urea synthesis and urea cycle-specific nitrogen flux would correlate with both phenotypic severity and carrier status in patients with a variety of different enzymatic deficiencies of the urea cycle. We studied control subjects, patients, and their relatives with different enzymatic deficiencies affecting the urea cycle while consuming a low protein diet. On a separate occasion the subjects either received a higher protein intake or were treated with an alternative route medication sodium phenylacetate/benzoate (Ucephan), or oral arginine supplementation. Total urea synthesis from all nitrogen sources was determined from [18O]urea labeling, and the utilization of peripheral nitrogen was estimated from the relative isotopic enrichments of [15N]urea and [15N]glutamine during i.v. co-infusions of [5-(amide)15N]glutamine and [18O]urea. The ratio of the isotopic enrichments of15N-urea/15N-glutamine distinguished normal control subjects (ratio = 0.42 ± 0.06) from urea cycle patients with late (0.17 ± 0.03) and neonatal (0.003 ± 0.007) presentations irrespective of enzymatic deficiency. This index of urea cycle activity also distinguished asymptomatic heterozygous carriers of argininosuccinate synthetase deficiency (0.22 ± 0.03), argininosuccinate lyase deficiency (0.35 ± 0.11), and partial ornithine transcarbamylase deficiency (0.26 ± 0.06) from normal controls. Administration of Ucephan lowered, and arginine increased, urea synthesis to the degree predicted from their respective rates of metabolism. The15N-urea/15N-glutamine ratio is a sensitive index ofin vivourea cycle activity and correlates with clinical severity. Urea synthesis is altered by alternative route medications and arginine supplementation to the degree that is to be expected from theory. This stable isotope protocol provides a sensitive tool for evaluating the efficacy of therapeutic modalities and acts as an aid to the diagnosis and management of urea cycle patients.
Variable β-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment
Authors: Brabletz Thomas , Jung Andreas , Reu Simone , Porzner Marc , Hlubek Falk , Kunz-Schughart Leoni A. , Knuechel Ruth , Kirchner Thomas
Published: July 2002

Invasion and dissemination of well-differentiated carcinomas are often associated with loss of epithelial differentiation and gain of mesenchyme-like capabilities of the tumor cells at the invasive front. However, when comparing central areas of primary colorectal carcinomas and corresponding metastases, we again found the same differentiated epithelial growth patterns. These characteristic phenotypic changes were associated with distinct expression patterns of β-catenin, the main oncogenic protein in colorectal carcinomas, and E-cadherin. Nuclear β-catenin was found in dedifferentiated mesenchyme-like tumor cells at the invasive front, but strikingly, as in central areas of the primary tumors, was localized to the membrane and cytoplasm in polarized epithelial tumor cells in the metastases. This expression pattern was accompanied by changes in E-cadherin expression and proliferative activity. On the basis of these data, we postulate that an important driving force for progression of well-differentiated colorectal carcinomas is the specific environment, initiating two transient phenotypic transition processes by modulating intracellular β-catenin distribution in tumor cells.
Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone
Authors: Halmos Gabor , Schally Andrew V. , Varga Jozsef L. , Plonowski Artur , Rekasi Zoltan , Czompoly Tamas
Published: July 2002

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancers
in vitro
and
in vivo
by mechanisms that include apparent direct effects through specific binding sites expressed on tumors and that differ from pituitary human GHRH (hGHRH) receptors. In this study, GHRH antagonist JV-1–38 (20 μg/day per animal s.c.) inhibited the growth of orthotopic CAKI-1 human renal cell carcinoma (RCC) by 83% and inhibited the development of metastases to lung and lymph nodes. Using ligand competition assays with
125
I-labeled GHRH antagonist JV-1–42, we demonstrated the presence of specific high-affinity (
K
d
= 0.25 ± 0.03 nM) binding sites for GHRH with a maximal binding capacity (
B
max
) of 70.2 ± 4.1 fmol/mg of membrane protein in CAKI-1 tumors. These receptors bind GHRH antagonists preferentially and display a lower affinity for hGHRH. The binding of
125
I-JV-1–42 is not inhibited by vasoactive intestinal peptide (VIP)-related peptides sharing structural homology with hGHRH. The receptors for GHRH antagonists on CAKI-1 tumors are distinct from binding sites detected with
125
I-VIP (
K
d
= 0.89 ± 0.14 nM;
B
max
= 183.5 ± 2.6 fmol/mg of protein) and also have different characteristics from GHRH receptors on rat pituitary as documented by the insignificant binding of [His
1
,
125
I-Tyr
10
,Nle
27
]hGHRH(1–32)NH
2
. Reverse transcription-PCR revealed the expression of splice variants of hGHRH receptor in CAKI-1 RCC. Biodistribution studies demonstrate an
in vivo
uptake of
125
I-JV-1–42 by the RCC tumor tissue. The presence of specific receptor proteins that bind GHRH antagonists in CAKI-1 RCC supports the view that distinct binding sites that mediate the inhibitory effect of GHRH antagonists are present on various human cancers.
Early human occupation of Western Europe: Paleomagnetic dates for two paleolithic sites in Spain
Authors: Oms O. , Parés J. M. , Martínez-Navarro B. , Agustí J. , Toro I. , Martínez-Fernández G. , Turq A.
Published: July 2002

The lacustrine deposits infilling the intramontane Guadix-Baza Basin, in the Betic Range of Southern Spain, have yielded abundant well-preserved lithic artifacts. In addition, the lake beds contain a wide range of micromammals includingMimomys saviniandAllophaiomys burgondiaeand large mammals such asMammuthusandHippopotamustogether with the African saber-toothed felidMegantereon. The association of the lithic artifacts along with the fossil assemblages, themselves of prime significance in the Eurasian mammal biochronology, is providing new insight into the controversy of the human settlement in Southern Europe. Despite the importance of the artifacts and fossil assemblage, estimates of the geological age of the site are still in conflict. Some attempts at dating the sediments have included biochronology, uranium series, amino acid racemization, and stratigraphic correlation with other well-dated sections in the basin, but so far have failed to yield unambiguous ages. Here we present paleomagnetic age dating at the relevant localities and thus provide useful age constraints for this critical paleoanthropological and mammal site. Our data provide firm evidence for human occupation in Southern Europe in the Lower Pleistocene, around 1 mega-annum ago. The current view of when and how hominids first dispersed into Europe needs to be reevaluated.
Modifications of retinal neurons in a mouse model of retinitis pigmentosa
Authors: Strettoi Enrica , Pignatelli Vincenzo
Published: July 2002

Animal models of retinitis pigmentosa include the rd mouse, in
which a mutation of a rod-specific phosphodiesterase leads to the rapid
loss of photoreceptors during the early postnatal life. Very little is
known about changes occurring in inner retinal neurons after
photoreceptor loss. These changes are important in view of the
possibility of restoring vision in retinas with photoreceptor
degeneration by means of cell transplantation or direct stimulation of
inner layers. In this paper, we show that bipolar and horizontal cells
of the rd mouse retina undergo dramatic morphological modifications
accompanying photoreceptor loss, demonstrating a dependence of second
order neurons on these cells. While describing modifications of the rd
retina, we also provide quantitative information about neurons of the
wild-type mouse retina, useful for future studies on genetically
altered animals.
Interference with
Pseudomonas
quinolone signal synthesis inhibits virulence factor expression by
Pseudomonas aeruginosa
Authors: Calfee M. Worth , Coleman James P. , Pesci Everett C.
Published: July 2002

Pseudomonas aeruginosa
is an opportunistic pathogen
that controls numerous virulence factors through intercellular signals.
This bacterium has two quorum-sensing systems (
las
and
rhl
), which act through the intercellular signals
N
-(3-oxododecanoyl)-
l
-homoserine lactone
(3-oxo-C
12
-HSL) and
N
-butyryl-
l
-homoserine lactone
(C
4
-HSL), respectively.
P. aeruginosa
also
produces a third intercellular signal that is involved in virulence
factor regulation. This signal, 2-heptyl-3-hydroxy-4-quinolone
[referred to as the
Pseudomonas
quinolone signal
(PQS)], is a secondary metabolite that is part of the
P.
aeruginosa
quorum-sensing hierarchy. PQS can induce both
lasB
(encodes LasB elastase) and
rhlI
(encodes the C
4
-HSL synthase) in
P.
aeruginosa
and is produced maximally during the late stationary
phase of growth. Because PQS is an intercellular signal that is part of
the quorum-sensing hierarchy and controls multiple virulence factors,
we began basic studies designed to elucidate its biosynthetic pathway.
First, we present data that strongly suggest that anthranilate is a
precursor for PQS.
P. aeruginosa
converted radiolabeled
anthranilate into radioactive PQS, which was bioactive. We also found
that an anthranilate analog (methyl anthranilate) would inhibit the
production of PQS. This analog was then shown to have a major negative
effect on elastase production by
P. aeruginosa
. These
data provide evidence that precursors of intercellular signals may
provide viable targets for the development of therapeutic treatments
that will reduce
P. aeruginosa
virulence.
Studies on the nonmevalonate pathway to terpenes: The role of the GcpE (IspG) protein
Authors: Hecht Stefan , Eisenreich Wolfgang , Adam Petra , Amslinger Sabine , Kis Klaus , Bacher Adelbert , Arigoni Duilio , Rohdich Felix
Published: July 2002

Recombinant
Escherichia coli
cells engineered for
the expression of the
xylB
gene in conjunction with
genes of the nonmevalonate pathway were supplied with
13
C-labeled 1-deoxy-
d
-xylulose. Cell extracts
were analyzed directly by NMR spectroscopy.
13
C-labeled
2
C
-methyl-
d
-erythritol 2,4-cyclodiphosphate
was detected at high levels in cells expressing
xylB
,
ispC
,
ispD
,
ispE,
and
ispF
. The additional expression of the
gcpE
gene afforded
1-hydroxy-2-methyl-2-(
E
)-butenyl 4-diphosphate as an
intermediate of the nonmevalonate pathway. Hypothetical mechanisms
involving conserved cysteine residues are proposed for the
enzymatic conversion of
2
C
-methyl-
d
-erythritol 2,4-cyclodiphosphate
into 1-hydroxy-2-methyl-2-(
E
)-butenyl 4-diphosphate
catalyzed by the GcpE protein.
Auxin modulates the degradation rate of Aux/IAA proteins
Authors: Zenser Nathan , Ellsmore Amanda , Leasure Colin , Callis Judy
Published: July 2002

Aux
/
IAA
gene family members were
first identified by their rapid transcriptional increase in response to
auxin. Auxin/indole-3-acetic acid protein (Aux/IAA) luciferase
(LUC) fusions expressed in
Arabidopsis
under control of
a non-auxin-responsive promoter were used to monitor the effect of
auxin on protein abundance independent of transcriptional regulation by
auxin. After 2 hr in the presence of 1 μM exogenous
dichlorophenoxyacetic acid (2,4D), a synthetic auxin, the levels of pea
IAA6 (PSIAA6) and
Arabidopsis
IAA1 LUC activity were
35% and 67%, respectively, of mock-treated genetically identical
seedlings, whereas the activity of LUC alone from equivalently treated
seedlings remained unaltered. The steady-state level of an Aux/IAA
fusion protein lacking domain II, one of the conserved domains found in
all Aux/IAA proteins, was not reduced in the presence of auxin.
Higher levels of exogenous auxin were required to affect the
steady-state level of the PSIAA6∷LUC fusion with a point mutation
in domain II. A 13-aa consensus sequence from domain II fused to LUC
created an auxin-responsive fusion protein. The change in steady-state
levels in response to auxin is extremely rapid, with a decrease in LUC
activity detectable by 2 min after auxin application. Direct half-life
measurements show that the decrease caused by exogenous auxin is due to
the decrease in fusion protein half-life. These results suggest that
auxin rapidly modulates the degradation rate of Aux/IAA proteins,
with higher levels of auxin increasing the proteolytic rate of
Aux/IAA family members.
Positive allosteric modulators of metabotropic glutamate 1 receptor: Characterization, mechanism of action, and binding site
Authors: Knoflach Frédéric , Mutel Vincent , Jolidon Synèse , Kew James N. C. , Malherbe Pari , Vieira Eric , Wichmann Jürgen , Kemp John A.
Published: July 2002

We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.
Genetically increased angiotensin I-converting enzyme level and renal complications in the diabetic mouse
Authors: Huang Wei , Gallois Yves , Bouby Nadine , Bruneval Patrick , Heudes Didier , Belair Marie-France , Krege John H. , Meneton Pierre , Marre Michel , Smithies Oliver , Alhenc-Gelas François
Published: July 2002

Diabetic nephropathy is a major risk factor for end-stage renal disease and cardiovascular diseases and has a marked genetic component. A common variant (D allele) of the
angiotensin Iconverting enzyme
(
ACE
) gene, determining higher enzyme levels, has been associated with diabetic nephropathy. To address causality underlying this association, we induced diabetes in mice having one, two, or three copies of the gene, normal blood pressure, and an enzyme level range (65–162% of wild type) comparable to that seen in humans. Twelve weeks later, the three-copy diabetic mice had increased blood pressures and overt proteinuria. Proteinuria was correlated to plasma ACE level in the three-copy diabetic mice. Thus, a modest genetic increase in ACE levels is sufficient to cause nephropathy in diabetic mice.
Caspase-1 and -3 are sequentially activated in motor neuron death in Cu,Zn superoxide dismutase-mediated familial amyotrophic lateral sclerosis
Authors: Pasinelli Piera , Houseweart Megan K. , Brown Robert H. , Cleveland Don W.
Published: July 2002

Familial amyotrophic lateral sclerosis-linked mutations in
copper-zinc superoxide dismutase cause motor neuron death through one
or more acquired toxic properties. An early event in the mechanism of
toxicity from such mutants is now demonstrated to be activation of
caspase-1. Neuronal death, however, follows only after months of
chronic caspase-1 activation concomitantly with activation of the
executioner caspase-3 as the final step in the toxic cascade. Thus, a
common toxicity of mutant SOD1 is a sequential activation of at least
two caspases, caspase-1 that acts slowly as a chronic initiator and
caspase-3 acting as the final effector of cell death.
Paternal relatedness and age proximity regulate social relationships among adult female rhesus macaques
Authors: Widdig Anja , Nürnberg Peter , Krawczak Michael , Streich Wolf Jürgen , Bercovitch Fred B.
Published: July 2002

Kin selection promotes the evolution of social behavior that increases the survival and reproductive success of close relatives. Among primates, maternal kinship frequently coincides with a higher frequency of grooming and agonistic aiding, but the extent to which paternal kinship influences adult female social relationships has not yet been investigated. Here, we examine the effect of both maternal and paternal kinship, as well as age proximity, on affiliative interactions among semifree-ranging adult female rhesus macaques,Macaca mulatta. Kinship was assessed by using both microsatellites and DNA-fingerprinting. Our study confirms that the closest affiliative relationships characterize maternal half-sisters. We provide evidence that adult females are significantly more affiliative with paternal half-sisters than with nonkin. Furthermore, paternal kin discrimination was more pronounced among peers than among nonpeers, indicating that age proximity has an additional regulatory effect on affiliative interactions. We propose that kin discrimination among cercopithecine primates emerges from ontogenetic processes that involve phenotype matching based on shared behavioral traits, such as inherited personality profiles, rather than physiological or physical characteristics.
Low amplification and fast visual pigment phosphorylation as mechanisms characterizing cone photoresponses
Authors: Tachibanaki Shuji , Tsushima Sawae , Kawamura Satoru
Published: July 2002

Vertebrate cone photoreceptors are known to show lower light sensitivity and briefer photoresponses than rod photoreceptors. To understand the molecular mechanisms characterizing cone photoresponses, we compared some of the reactions in the phototransduction cascade between rods and cones. For this purpose, rods and cones were obtained in quantities large enough to do biochemical studies. The cells were purified from the retina of carp (Cyprinus carpio) with a stepwise Percoll gradient. The purified rod fraction contained almost no other kinds of cells besides rods, and the purified cone fraction contained a mixture of red-, green-, and blue-sensitive cones in the ratio 3:≈1:≈1. We prepared membrane preparations from the rod and the cone fraction, and in these membranes, we measured activation efficiencies of the reactions in the phototransduction cascade. The results showed that the signal amplification is lower in the cone membranes, which accounts for the lower light sensitivity in cones. Furthermore, we measured the time courses of visual pigment phosphorylation. The result showed that the phosphorylation is much faster in the cone membranes, which also explains the lower light sensitivity and, in addition, the briefer photoresponse in cones.
Prion infection impairs the cellular response to oxidative stress
Authors: Milhavet Ollivier , McMahon Hilary E. M. , Rachidi Walid , Nishida Noriyuki , Katamine Shigeru , Mangé Alain , Arlotto Marie , Casanova Danielle , Riondel Jacqueline , Favier Alain , Lehmann Sylvain
Published: July 2002

The molecular mechanism of neurodegeneration in transmissible
spongiform encephalopathies remains uncertain. In this study, it was
demonstrated that prion-infected hypothalamic neuronal GT1 cells
displayed a higher sensitivity to induced oxidative stress over
noninfected cells. In addition, the infected cells presented an
increased lipid peroxidation and signs of apoptosis associated
with a dramatic reduction in the activities of the
glutathione-dependent and superoxide dismutase antioxidant systems.
This study indicates for the first time that prion infection results in
an alteration of the molecular mechanisms promoting cellular resistance
to reactive oxygen species. This finding is vital for future
therapeutic approaches in transmissible spongiform encephalopathies and
the understanding of the function of the prion protein.
Powerful and prolonged protection of human retinal pigment epithelial cells, keratinocytes, and mouse leukemia cells against oxidative damage: The indirect antioxidant effects of sulforaphane
Authors: Gao Xiangqun , Dinkova-Kostova Albena T. , Talalay Paul
Published: July 2002

Mammalian cells are equipped with elaborate systems
for protection against the toxicity of reactive oxygen and nitrogen
species and electrophiles that are constant dangers to the integrity
of their DNA. Phase 2 enzymes (e.g., glutathione
transferases, NAD(P)H:quinone reductase) and glutathione synthesis are
widely recognized as playing major protective roles against
electrophilic carcinogens, but their antioxidant functions have
attracted far less attention. The cytotoxicities of four oxidative
stressors (menadione,
tert
-butyl hydroperoxide,
4-hydroxynonenal, and peroxynitrite) for human adult retinal pigment
epithelial cells (ARPE-19) were quantified by measuring the
concentration dependence of cell death and were expressed as the median
effect dose (
D
m
) for each oxidant. After
treatment of ARPE-19 cells for 24 h with 0–5 μM concentrations
of sulforaphane (the powerful Phase 2 enzyme inducer isolated from
broccoli), the toxicities of the oxidants were markedly reduced as
shown by 1.5- to 3-fold increases in
D
m
values. The magnitude of protection was a function of the nature of the
oxidants and the concentrations of both the oxidants and sulforaphane.
Protection was prolonged and persisted for several days after removal
of sulforaphane before returning to control levels. The
sulforaphane-dependent increases in specific activities of cytosolic
quinone reductase and the glutathione levels were highly significantly
correlated with the degree of protection as measured by
D
m
values. Antioxidant protection was also
demonstrated for human HaCaT keratinocytes and L1210 murine leukemia
cells. It is therefore highly likely that the multifaceted and
prolonged antioxidant protection provided by sulforaphane is a general
phenomenon that is mediated through induction of the Phase 2
enzyme response.
Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae.
Authors: Davidson J F , Whyte B , Bissinger P H , Schiestl R H
Published: July 2002

The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxidant genes catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the lethal effect of heat than isogenic wild-type cells. Overexpression of catalase and superoxide dismutase genes caused an increase in thermotolerance. Anaerobic conditions caused a 500- to 20,000-fold increase in thermotolerance. The thermotolerance of cells in anaerobic conditions was immediately abolished upon oxygen exposure. HSP104 is not responsible for the increased resistance of anaerobically grown cells. The thermotolerance of anaerobically grown cells is not due to expression of heat-shock proteins. By using an oxidation-dependent fluorescent molecular probe a 2- to 3-fold increase in fluorescence was found upon heating. Thus, we conclude that oxidative stress is involved in heat-induced cell death.
The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors.
Authors: Andreansky S S , He B , Gillespie G Y , Soroceanu L , Markert J , Chou J , Roizman B , Whitley R J
Published: July 2002

Due to lack of effective therapy, primary brain tumors are the focus of intense investigation of novel experimental approaches that use vectors and recombinant viruses. Therapeutic approaches have been both indirect, whereby vectors are used, or direct to allow for direct cell killing by the introduced virus. Genetically engineered herpes simplex viruses are currently being evaluated as an experimental approach to eradicate malignant human gliomas. Initial studies with gamma (1)34.5 mutants, R3616 (from which both copies of the gamma (1)34.5 gene have been deleted) and R4009 (a construct with two stop codons inserted into the gamma (1)34.5 gene), have been assessed. In a syngeneic scid mouse intracranial tumor model, recombinant herpes simplex virus can be experimentally used for the treatment of brain tumors. These viruses and additional engineered viruses were subsequently tested in human glioma cells both in vitro and in vivo. Using a xenogeneic scid mouse intracranial glioma model, R4009 therapy of established tumors significantly prolonged survival. Most importantly, long-term survival was achieved, with histologic evidence that R4009 eradicated intracranial tumors in this model. Furthermore, the opportunity to evaluate gamma (1)34.5 mutants that have enhanced oncolytic activity, e.g., R8309 where the carboxyl terminus of the gamma (1)34.5 gene has been replaced by the murine homologue, MyD116, are considered.