bluuebunny/crossref_metadata_2025 huggingface dataset:
Wavelet bicoherence analysis of strong plasma turbulence at the Earth’s quasiparallel bow shock
Authors: Dudok de Wit T. , Krasnosel’skikh V. V.
Published: July 2002

Nonlinear interactions in strong plasma turbulence have been investigated in the vicinity of the Earth’s quasiparallel bow shock by making use of magnetometer data from the AMPTE–UKS (Active Magnetospheric Particle Tracer Explorers–United Kingdom Subsatellite) spacecraft. Using a new and more robust estimator of the bicoherence, it is shown that the large-amplitude monolithic magnetic structures observed upstream the shock front interact with the whistler wave trains that accompany them. A unified picture emerges, in which the whistlers progressively grow from these soliton-like structures through nonlinear interaction, similarly to the dynamical evolution of step-like profiles in the Korteweg–de Vries equation.
Hard x-ray emission in high intensity femtosecond laser–target interaction
Authors: Yu J. , Jiang Z. , Kieffer J. C. , Krol A.
Published: July 2002

Hard x-ray spectra (10–100 keV) created in high contrast, 400 fs, laser pulse interaction with solid targets, have been studied for laser intensities in the 1017–1019 W/cm2 range. The target atomic numbers (Z) extended from Z=13 to Z=73. The measured conversion efficiency at Ag Kα emission line was 10−3% at 5×1018 W/cm2. It has been confirmed that the hot electron temperature increased as (Iλ2)1/3 and the fraction of laser energy in hot electrons follows scaling law of (Iλ2)3/4.
Evaluation of 1/ν neoclassical transport in stellarators
Authors: Nemov V. V. , Kasilov S. V. , Kernbichler W. , Heyn M. F.
Published: July 2002

Using an analytic solution of the kinetic equation in the 1/ν regime, a new formula for the neoclassical transport coefficients is obtained which takes into account all classes of trapped particles. This formula holds in any coordinate system and no simplifying assumptions about the magnetic field are needed. Therefore it is also applicable to complex magnetic fields given in real space coordinates. The method and the results can be used to optimize magnetic field configurations with respect to the 1/ν regime. The method is bench-marked against Monte Carlo calculation both for the l=3 classical stellarator model and also for the original Helias configuration [J. Nührenberg and R. Zille, Phys. Lett. A 114, 129 (1986)] with a more complex magnetic field structure. Some features of transport for Helias are clarified by analyzing the bounce-averaged drift velocity.
Gain measurements near 351 nm in 3He/Xe/NF3 mixtures excited by fragments from the 3He(n, p) 3H reaction
Authors: Hays G. N. , McArthur D. A. , Neal D. R. , Rice J. K.
Published: July 2002

We report direct gain measurements on the 351-nm XeF(B–X) transition in mixtures of 3He/Xe/NF3, pumped with moderated neutrons produced in a fast-burst reactor. The peak small-signal gain was 0.7%/cm at a deposited power of 5 kW/cc in the gas.
Pseudomorphic In0.53Ga0.47As/AlAs/InAs resonant tunneling diodes with peak-to-valley current ratios of 30 at room temperature
Authors: Broekaert Tom P. E. , Lee Wai , Fonstad Clifton G.
Published: July 2002

Pseudomorphic In0.53 Ga0.47 As/AlAs/InAs resonant tunneling diodes have been grown on InP substrates by molecular beam epitaxy. Peak-to-valley current ratios as high as 30 at 300 K and 63 at 77 K are obtained on a structure with barriers of ten atomic layers AlAs, and a well consisting of three atomic layers of In0.53 Ga0.47 As, six atomic layers of InAs, and three atomic layers of In0.53 Ga0.47 As. For comparison pseudomorphic In0.53 Ga0.47 As/AlAs with In0.53 Ga0.47 As well structures have also been fabricated. For the In0.53 Ga0.47 As well structures, peak-to-valley current ratios as high as 23 have been obtained at 300 K, and, in other devices with lower current densities, two resonances are observed at room temperature.
β-Amyloid precursor protein transgenic mice that harbor diffuse Aβ deposits but do not form plaques show increased ischemic vulnerability: Role of inflammation
Authors: Koistinaho Milla , Kettunen Mikko I. , Goldsteins Gundars , Keinänen Riitta , Salminen Antero , Ort Michael , Bures Jan , Liu David , Kauppinen Risto A. , Higgins Linda S. , Koistinaho Jari
Published: July 2002

β-amyloid (Aβ), derived form the β-amyloid precursor protein (APP), is important for the pathogenesis of Alzheimer's disease (AD), which is characterized by progressive decline of cognitive functions, formation of Aβ plaques and neurofibrillary tangles, and loss of neurons. However, introducing a human wild-type or mutant APP gene to rodent models of AD does not result in clear neurodegeneration, suggesting that contributory factors lowering the threshold of neuronal death may be present in AD. Because brain ischemia has recently been recognized to contribute to the pathogenesis of AD, we studied the effect of focal brain ischemia in 8- and 20-month-old mice overexpressing the 751-amino acid isoform of human APP. We found that APP751 mice have higher activity of p38 mitogen-activated protein kinase (p38 MAPK) in microglia, the main immune effector cells within the brain, and increased vulnerability to brain ischemia when compared with age-matched wild-type mice. These characteristics are associated with enhanced microglial activation and inflammation but not with altered regulation of cerebral blood flow, as assessed by MRI and laser Doppler flowmetry. Suppression of inflammation with aspirin or inhibition of p38 MAPK with a selective inhibitor, SD-282, abolishes the increased neuronal vulnerability in APP751 transgenic mice. SD-282 also suppresses the expression of inducible nitric-oxide synthase and the binding activity of activator protein 1. These findings elucidate molecular mechanisms of neuronal injury in AD and suggest that antiinflammatory compounds preventing activation of p38 MAPK in microglia may reduce neuronal vulnerability in AD.
Learning improves growth rate in grasshoppers
Authors: Dukas Reuven , Bernays Elizabeth A.
Published: July 2002

To quantify the adaptive significance of insect learning, we
documented the behavior and growth rate of grasshoppers
(
Schistocerca americana
) in an
environment containing two artificial food types, one providing a
balanced diet of protein and carbohydrate, which maximizes growth, and
the other being carbohydrate-deficient, which is unsuitable for growth.
Grasshoppers in the Learning treatment experienced a predictable
environment, where the spatial location, taste, and color of each food
source remained constant throughout the experiment. In contrast,
grasshoppers of the Random treatment developed in a temporally varying
environment, where the spatial location, taste, and color of the
balanced and deficient food types randomly alternated twice each day.
Our results show that the grasshoppers that could employ associative
learning for diet choice experienced higher growth rates than
individuals of the Random treatment, demonstrating the adaptive
significance of learning in a small short-lived insect.
Poly(ADP-ribose) polymerase-1 dependence of stress-induced transcription factors and associated gene expression in glia
Authors: Ha Hyo Chol , Hester Lynda D. , Snyder Solomon H.
Published: July 2002

Poly(ADP-ribose) polymerase-1 (PARP-1, EC
2.4.2.30
), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Excessive activation of PARP-1 by cellular insults depletes its substrate β-nicotinamide adenine dinucleotide and ATP, leading to cell death. PARP-1-deficient (PARP-1
−/−
) mice are protected from several forms of inflammation. In the present study, we demonstrate in PARP-1
−/−
glial cells a loss of several stress-activated transcription factors as well as decreased expression of genes for cytokines and cellular adhesion molecules. We also show that augmented expression of some of these genes is independent of PARP-1 catalytic activity. These findings indicate that PARP-1 plays a pivotal role in the initial inflammatory response by modulating transcription of inflammation-linked genes.
Targeted point mutations of
p53
lead to dominant-negative inhibition of wild-type p53 function
Authors: de Vries Annemieke , Flores Elsa R. , Miranda Barbara , Hsieh Harn-Mei , van Oostrom Conny Th. M. , Sage Julien , Jacks Tyler
Published: July 2002

The
p53
tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line
p53
mutations cause a familial predisposition for cancer. Germ-line or sporadic
p53
mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to γ irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in
p53
-dependent apoptotic pathways after treatment with γ irradiation or doxorubicin, whereas
p53
-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated
p53
can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.
In vivo
NMR microscopy allows short-term serial assessment of multiple skeletal implications of corticosteroid exposure
Authors: Takahashi Masaya , Wehrli Felix W. , Hilaire Luna , Zemel Babette S. , Hwang Scott N.
Published: July 2002

Corticosteroids are in widespread clinical use but are known to have adverse skeletal side effects. Moreover, it is not known how soon these effects become apparent. Here, we describe a longitudinal approach to evaluate the short-term implications of excess corticosteroid exposure by quantitative
in vivo
magnetic resonance imaging and spectroscopy in conjunction with digital image processing and analysis in a rabbit model. Two-week treatment with dexamethasone induced a significant reduction in trabecular bone volume, which occurred at the expense of uniform trabecular thinning without affecting network architecture. Paralleling the loss in bone volume was conversion of hematopoietic to yellow marrow in the femoral metaphysis and atrophy of the femoral epiphyseal growth plate. This work demonstrates that detailed quantitative morphometric and physiological information can be obtained noninvasively at multiple skeletal locations. The method is likely to eventually replace invasive histomorphometry in that it obviates the need to sacrifice groups of animals at multiple time points. Finally, this work, which was performed on a clinical scanner, has implications for evaluating patients on high-dose steroid treatment.
Recovery of
Diadema antillarum
reduces macroalgal cover and increases abundance of juvenile corals on a Caribbean reef
Authors: Edmunds Peter J. , Carpenter Robert C.
Published: July 2002

The transition of many Caribbean reefs from coral to macroalgal
dominance has been a prominent issue in coral reef ecology for more
than 20 years. Alternative stable state theory predicts that these
changes are reversible but, to date, there is little indication of this
having occurred. Here we present evidence of the initiation of such a
reversal in Jamaica, where shallow reefs at five sites along 8 km of
coastline now are characterized by a sea urchin-grazed zone with a mean
width of 60 m. In comparison to the seaward algal zone, macroalgae
are rare in the urchin zone, where the density of
Diadema
antillarum
is 10 times higher and the density of juvenile
corals is up to 11 times higher. These densities are close to those
recorded in the late 1970s and early 1980s and are in striking contrast
to the decade-long recruitment failure for both
Diadema
and scleractinians. If these trends continue and expand spatially,
reefs throughout the Caribbean may again become dominated by corals and
algal turf.
Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases
Authors: Robker R. L. , Russell D. L. , Espey L. L. , Lydon J. P. , O'Malley B. W. , Richards J. S.
Published: July 2002

Ovulation is a precisely timed process by which a mature oocyte is released from an ovarian follicle. This process is initiated by the pituitary surge of luteinizing hormone (LH), is temporally associated with transcriptional regulation of numerous genes, and is presumed to involve the synthesis and/or activation of specific proteases that degrade the follicle wall. The progesterone receptor (PR), a nuclear receptor transcription factor, is induced in granulosa cells of preovulatory follicles in response to the LH surge and has been shown to be essential for ovulation, because mice lacking PR fail to ovulate and are infertile. Using these mice as a model in which to elucidate PR-regulated genes in the ovulation process, we show that the matrix metalloproteinases MMP-2 and MMP-9 are not targets of PR during ovulation. In contrast, two other proteases, ADAMTS-1 (A disintegrin and metalloproteinase with thrombospondin-like motifs) and cathepsin L (a lysosomal cysteine protease), are transcriptional targets of PR action. ADAMTS-1 is induced after LH stimulation in granulosa cells of preovulatory follicles and depends on PR. Cathepsin L is induced in granulosa cells of growing follicles by follicle-stimulating hormone, but the highest levels of cathepsin L mRNA occur in preovulatory follicles in response to LH in a PR-dependent manner. The identification of two regulated proteases in the ovary, together with their abnormal expression in anovulatory PR knockout mice, suggests that each plays a critical role in follicular rupture and represents a major advance in our understanding of the proteolytic events that control ovulation.
Carcinogen-specific induction of genetic instability
Authors: Bardelli Alberto , Cahill Daniel P. , Lederer Gabi , Speicher Michael R. , Kinzler Kenneth W. , Vogelstein Bert , Lengauer Christoph
Published: July 2002

It has been proposed recently that the type of genetic instability
in cancer cells reflects the selection pressures exerted by specific
carcinogens. We have tested this hypothesis by treating immortal,
genetically stable human cells with representative carcinogens. We
found that cells resistant to the bulky-adduct-forming agent
2-amino-1-methyl-6-phenylimidazo[4,5-
b
]pyridine
(PhIP) exhibited a chromosomal instability (CIN), whereas cells
resistant to the methylating agent
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine
(MNNG) exhibited a microsatellite instability (MIN) associated with
mismatch repair defects. Conversely, we found that cells purposely made
into CIN cells are resistant to PhIP, whereas MIN cells are resistant
to MNNG. These data demonstrate that exposure to specific carcinogens
can indeed select for tumor cells with distinct forms of genetic
instability and
vice versa
.
Circular dichroism readout of sugar recognition in the cleft of a fused-pyridine receptor
Authors: Tamaru Shun-ichi , Shinkai Seiji , Khasanov Alisher B. , Bell Thomas W.
Published: July 2002

Dicarboxamide host 2 forms 1:1 complexes with
n
-octyl pyranosides derived from
d
-glucose,
d
-mannose,
d
-galactose,
d
-fucose,
d
-lyxose, and
d
-arabinose. Association constants (
K
a
) in the range of 77–940 M
−1
were measured in chloroform by means of induced circular dichroism and fluorescence spectroscopy. Variations in
K
a
values correspond qualitatively to expected differences in hydrogen-bonding abilities of guest hydroxyl groups. Induced circular dichroism effects for complexes of saccharides bearing equatorial 3-OH, 4-OH, and 6-OH groups show that the host chromophore is twisted in a
P
-helical conformation. A structural model is proposed that is also consistent with the results of previous studies involving complexation of dicarboxylic acid 1 with cationic saccharides in methanol.
Colocalization and coassembly of two human brain M-type potassium channel subunits that are mutated in epilepsy
Authors: Cooper Edward C. , Aldape Kenneth D. , Abosch Aviva , Barbaro Nicholas M. , Berger Mitchel S. , Peacock Warwick S. , Jan Yuh Nung , Jan Lily Yeh
Published: July 2002

Acetylcholine excites many central and autonomic neurons through inhibition of M-channels, slowly activating, noninactivating voltage-gated potassium channels. We here provide information regarding the
in vivo
distribution and biochemical characteristics of human brain KCNQ2 and KCNQ3, two channel subunits that form M-channels when expressed
in vitro
, and, when mutated, cause the dominantly inherited epileptic syndrome, benign neonatal familial convulsions. KCNQ2 and KCNQ3 proteins are colocalized in a somatodendritic pattern on pyramidal and polymorphic neurons in the human cortex and hippocampus. Immunoreactivity for KCNQ2, but not KCNQ3, is also prominent in some terminal fields, suggesting a presynaptic role for a distinct subgroup of M-channels in the regulation of action potential propagation and neurotransmitter release. KCNQ2 and KCNQ3 can be coimmunoprecipitated from brain lysates. Further, KCNQ2 and KCNQ3 are coassociated with tubulin and protein kinase A within a Triton X-100-insoluble protein complex. This complex is not associated with low-density membrane rafts or with
N
-methyl-
d
-aspartate receptors, PSD-95 scaffolding proteins, or other potassium channels tested. Our studies thus provide a view of a signaling complex that may be important for cognitive function as well as epilepsy. Analysis of this complex may shed light on the unknown transduction pathway linking muscarinic acetylcholine receptor activation to M-channel inhibition.
Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase
Authors: Tang Pei-Zhong , Tsai-Morris Chon-Hwa , Dufau Maria L.
Published: July 2002

A previously unidentified gonadotropin-regulated long chain
acyl-CoA synthetase (GR-LACS) was cloned and characterized as a 79-kDa
cytoplasmic protein expressed in Leydig cells of the rat testis.
GR-LACS shares sequence identity with two conserved regions of the LACS
and luciferase families, including the ATP/AMP binding domain and the
25-aa fatty acyl-CoA synthetase signature motif, but displays low
overall amino acid similarities (23–28%). GR-LACS mRNA is expressed
abundantly in Leydig cells of the adult testis and to a lesser degree
in the seminiferous tubules in spermatogonia and Sertoli cells. It is
also observed in ovary and brain. Immunoreactive protein expression was
observed mainly in Leydig cells and minimally in the tubules but was
not detected in other tissues.
In vivo
, treatment with a
desensitizing dose of human chorionic gonadotropin caused
transcriptional down-regulation of GR-LACS expression in Leydig cells.
The expressed protein present in the cytoplasm of transfected cells
displayed acyl-CoA synthetase activity for long chain fatty acid
substrates. GR-LACS may contribute to the provision of energy
requirements and to the biosynthesis of steroid precursors and could
participate through acyl-CoA's multiple functions in the regulation of
the male gonad.
Maternal effects and the evolution of aposematic signals
Authors: Brodie Edmund D. , Agrawal Aneil F.
Published: July 2002

Aposematic signals that warn predators of the noxious qualities of prey gain their greatest selective advantage when predators have already experienced similar signals. Existing theory explains how such signals can spread through selective advantage after they are present at some critical frequency, but is unclear about how warning signals can be selectively advantageous when the trait is initially rare (i.e., when it first arises through mutation) and predators are naive. When aposematism is controlled by a maternal effect gene, the difficulty of initial rarity may be overcome. Unlike a zygotically expressed gene, a maternally expressed aposematism gene will be hidden from selection because it is not phenotypically expressed in the first individual with the mutation. Furthermore, the first individual carrying the new mutation will produce an entire family of aposematic offspring, thereby providing an immediate fitness advantage to this gene.
Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons
Authors: Huang Yadong , Liu Xiao Qin , Wyss-Coray Tony , Brecht Walter J. , Sanan David A. , Mahley Robert W.
Published: July 2002

Human apolipoprotein (apo) E4, a major risk factor for Alzheimer's disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(Δ272–299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.
Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization
Authors: Gómez-Moutón Concepción , Abad Jose Luis , Mira Emilia , Lacalle Rosa Ana , Gallardo Eduard , Jiménez-Baranda Sonia , Illa Isabel , Bernad Antonio , Mañes Santos , Martínez-A. Carlos
Published: July 2002

Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction. Our data suggest that raft partitioning is a major determinant for protein redistribution in polarized T cells, as ectopic expression of raft-associated proteins results in their asymmetric redistribution, whereas non-raft-partitioned mutants of these proteins are distributed homogeneously in the polarized cell membrane. Both acquisition of a migratory phenotype and SDF-1α-induced chemotaxis are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft redistribution requires an intact actin cytoskeleton, but is insensitive to microtubule disruption. We propose that membrane protein segregation not only between raft and nonraft domains but also between distinct raft subdomains may be an organizational principle that mediates redistribution of specialized molecules needed for T cell migration.
The proliferative and antiapoptotic effects of substance P are facilitated by formation of a β-arrestin-dependent scaffolding complex
Authors: DeFea K. A. , Vaughn Z. D. , O'Bryan E. M. , Nishijima D. , Déry O. , Bunnett N. W.
Published: July 2002

A requirement for scaffolding complexes containing internalized G protein-coupled receptors and β-arrestins in the activation and subcellular localization of extracellular signal-regulated kinases 1 and 2 (ERK1/2) has recently been proposed. However, the composition of these complexes and the importance of this requirement for function of ERK1/2 appear to differ between receptors. Here we report that substance P (SP) activation of neurokinin-1 receptor (NK1R) stimulates the formation of a scaffolding complex comprising internalized receptor, β-arrestin, src, and ERK1/2 (detected by gel filtration, immunoprecipitation, and immunofluorescence). Inhibition of complex formation, by expression of dominant-negative β-arrestin or a truncated NK1R that fails to interact with β-arrestin, inhibits both SP-stimulated endocytosis of the NK1R and activation of ERK1/2, which is required for the proliferative and antiapoptotic effects of SP. Thus, formation of a β-arrestin-containing complex facilitates the proliferative and antiapoptotic effects of SP, and these effects of SP could be diminished in cells expressing truncated NK1R corresponding to a naturally occurring variant.
Hippocampal acetylcholine release during memory testing in rats: augmentation by glucose.
Authors: Ragozzino M E , Unick K E , Gold P E
Published: July 2002

Several lines of evidence indicate that a modest increase in circulating glucose levels enhances memory. One mechanism underlying glucose effects on memory may be an increase in acetylcholine (ACh) release. The present experiment determined whether enhancement of spontaneous alternation performance by systemic glucose treatment is related to an increase in hippocampal ACh output. Samples of extracellular ACh were assessed at 12-min intervals using in vivo microdialysis with HPLC-EC. Twenty-four minutes after an intraperitoneal injection of saline or glucose (100, 250, or 1000 mg/kg), rats were tested in a four-arm cross maze for spontaneous alternation behavior combined with microdialysis collection. Glucose at 250 mg/kg, but not 100 or 1000 mg/kg, produced an increase in spontaneous alternation scores (69.5%) and ACh output (121.5% versus baseline) compared to alternation scores (44.7%) and ACh output (58.9% versus baseline) of saline controls. The glucose-induced increase in alternation scores and ACh output was not secondary to changes in locomotor activity. Saline and glucose (100-1000 mg/kg) treatment had no effect on hippocampal ACh output when rats remained in the holding chamber. These findings suggest that glucose may enhance memory by directly or indirectly increasing the release of ACh. The results also indicate that hippocampal ACh release is increased in rats performing a spatial task. Moreover, because glucose enhanced ACh output only during behavioral testing, circulating glucose may modulate ACh release only under conditions in which cholinergic cells are activated.
Influence of cisplatin intrastrand crosslinking on the conformation, thermal stability, and energetics of a 20-mer DNA duplex.
Authors: Poklar N , Pilch D S , Lippard S J , Redding E A , Dunham S U , Breslauer K J
Published: July 2002

cis-Diamminedichloroplatinum(II) (cisplatin) is a widely used anticancer drug that binds to and crosslinks DNA. The major DNA adduct of the drug results from coordination of two adjacent guanine bases to platinum to form the intrastrand crosslink cis-[Pt(NH3)2[d(GpG)-N7(1), -N7(2)]] (cis-Pt-GG). In the present study, spectroscopic and calorimetric techniques were employed to characterize the influence of this crosslink on the conformation, thermal stability, and energetics of a site-specifically platinated 20-mer DNA duplex. CD spectroscopic and thermal denaturation data revealed that the crosslink alters the structure of the host duplex, consistent with a shift from a B-like to an A-like conformation; lowers its thermal stability by approximately 9 degrees C; and reduces its thermodynamic stability by 6.3 kcal/mol at 25 degrees C, most of which is enthalpic in origin; but it does not alter the two-state melting behavior exhibited by the parent, unmodified duplex, despite the significant crosslink-induced changes noted above. The energetic consequences of the cis-Pt-GG crosslink are discussed in relation to the structural perturbations it induces in DNA and to how these crosslink-induced perturbations might modulate protein binding.
A plastid enzyme arrested in the step of precursor translocation in vivo.
Authors: Reinbothe S , Reinbothe C , Neumann D , Apel K
Published: July 2002

The key enzyme of chlorophyll biosynthesis in higher plants, NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR, EC 1.3.1.33), accumulates in its precursor form (pPORA) in barley. pPORA is bound to the chloroplasts and is able to interact with the enzyme's substrate, Pchlide, at both the cytosolic as well as the stromal side of the plastid envelope. The interaction with intraplastidic Pchlide, formed in ATP-containing chloroplasts upon feeding with -aminolevulinic acid, drives vectorial translocation of pPORA across the plastid envelope membranes. In contrast, exogenously applied Pchlide causes the release of the envelope-bound precursor protein to the cytosol. Both processes compete with each other if intra- and extraplastidic Pchlide are applied simultaneously. A cytosolic heat shock cognate protein of Mr 70,000 present in wheat germ and barley leaf protein extracts appears to prevent the release of the pPORA to the cytosol in vivo, however.
myb
provides an essential function during
Drosophila
development
Authors: Katzen Alisa L. , Bishop J. Michael
Published: July 2002

Vertebrate c-
myb
encodes a transcription factor
thought to play an important role in the cell cycle. To gain further
insight into
myb
function, we have been studying a
related gene in
Drosophila
. We found that
Drosophila myb
is abundantly expressed throughout
development in all mitotically active tissues, at lower levels in some
postmitotic tissues, but not at detectable levels in polyploid larval
tissues. We performed genetic screens to isolate recessive lethal
mutations in the chromosomal region that includes the
myb
gene. We obtained two temperature-sensitive alleles
of
myb
, demonstrating that the gene provides an
essential function. Examination of the mutant phenotype revealed that
Drosophila myb
is important for both embryonic and
imaginal development and that
myb
serves a role in the
development of many tissues and during oogenesis.
A novel mechanism of action of tetracyclines: Effects on nitric oxide synthases
Authors: Amin Ashok R. , Attur Mukundan G. , Thakker Geeta D. , Patel Prakash D. , Vyas Pranav R. , Patel Rajesh N. , Patel Indravadan R. , Abramson Steven B.
Published: July 2002

Tetracyclines have recently been shown to have “chondroprotective” effects in inflammatory arthritides in animal models. Since nitric oxide (NO) is spontaneously released from human cartilage affected by osteoarthritis (OA) or rheumatoid arthritis in quantities sufficient to cause cartilage damage, we evaluated the effect of tetracyclines on the expression and function of human OA-affected nitric oxide synthase (OA-NOS) and rodent inducible NOS (iNOS). Among the tetracycline group of compounds, doxycycline > minocycline blocked and reversed both spontaneous and interleukin 1β-induced OA-NOS activity inex vivoconditions. Similarly, minocycline ≥ doxycycline inhibited both lipopolysaccharide- and interferon-γ-stimulated iNOS in RAW 264.7 cellsin vitro, as assessed by nitrite accumulation. Although both these enzyme isoforms could be inhibited by doxycycline and minocycline, their susceptibility to each of these drugs was distinct. Unlike acetylating agents or competitive inhibitors ofl-arginine that directly inhibit the specific activity of NOS, doxycycline or minocycline has no significant effect on the specific activity of iNOS in cell-free extracts. The mechanism of action of these drugs on murine iNOS expression was found to be, at least in part, at the level of RNA expression and translation of the enzyme, which would account for the decreased iNOS protein and activity of the enzyme. Tetracyclines had no significant effect on the levels of mRNA for β-actin and glyceraldehyde-3-phosphate dehydrogenase nor on levels of protein of β-actin and cyclooxygenase 2 expression. These studies indicate that a novel mechanism of action of tetracyclines is to inhibit the expression of NOS. Since the overproduction of NO has been implicated in the pathogenesis of arthritis, as well as other inflammatory diseases, these observations suggest that tetracyclines should be evaluated as potential therapeutic modulators of NO for various pathological conditions.
C factor, a cell-surface-associated intercellular signaling protein, stimulates the cytoplasmic Frz signal transduction system in Myxococcus xanthus.
Authors: Søgaard-Andersen L , Kaiser D
Published: July 2002

C factor, an intercellular signaling protein, is required for aggregation and sporulation of the social bacterium, Myxococcus xanthus. We report that C factor, which normally is associated with the cell surface, provides input to the Frz signal transduction cascade. Elements of this cascade have sequence homology to bacterial chemotaxis systems and are known to control the frequency of gliding reversal. Exposure of developing cells of a C-factor-less mutant (csgA) to purified C factor increases the ratio of methylated to nonmethylated FrzCD protein, the Frz homolog of the methyl-accepting chemotaxis proteins. Methylation depends on the cognate methyltransferase FrzF, and its extent increases with the concentration of C factor. C-factor-induced methylation also depends on the product of a gene, called class II, which is necessary in vivo for all known responses to C factor. A model for aggregation is proposed in which C factor stimulates the Frz cascade and thereby decreases cell reversals in a way that preferentially leads cells into an aggregate.
Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells.
Authors: Pulaski B A , Yeh K Y , Shastri N , Maltby K M , Penney D P , Lord E M , Frelinger J G
Published: July 2002

We show that interleukin 3 (IL-3) enhances the generation of tumor-specific cytotoxic T lymphocytes (CTLs) through the stimulation of host antigen-presenting cells (APCs). The BALB/c (H-2d) spontaneous lung carcinoma line 1 was modified by gene transfection to express ovalbumin as a nominal "tumor antigen" and to secrete IL-3, a cytokine enhancing myeloid development. IL-3-transfected tumor cells are less tumorigenic than the parental cell line, and tumor-infiltrating lymphocytes isolated from these tumors contain increased numbers of tumor-specific CTLs. By using B3Z86/90.14 (B3Z), a unique T-cell hybridoma system restricted to ovalbumin/H-2b and implanting the tumors in (BALB/c x C57BL/6)F1 (H-2d/b) mice, we demonstrate that the IL-3-transfected tumors contain an increased number of a rare population of host cells that can process and "re-present" tumor antigen to CTLs. Electron microscopy allowed direct visualization of these host APCs, and these studies, along with surface marker phenotyping, indicate that these APCs are macrophage-like. The identification of these cells and their enhancement by IL-3 offers a new opportunity for tumor immunotherapy.
Protein folding: How the mechanism of GroEL action is defined by kinetics
Authors: Frieden Carl , Clark A. Clay
Published: July 2002

We propose a mechanism for the role of the bacterial chaperonin GroEL in folding proteins. The principal assumptions of the mechanism are (
i
) that many unfolded proteins bind to GroEL because GroEL preferentially binds small unstructured regions of the substrate protein, (
ii
) that substrate protein within the cavity of GroEL folds by the same kinetic mechanism and rate processes as in bulk solution, (
iii
) that stable or transient complexes with GroEL during the folding process are defined by a kinetic partitioning between formation and dissociation of the complex and the rate of folding and unfolding of the protein, and (
iv
) that dissociation from the complex in early stages of folding may lead to aggregation but dissociation at a late stage leads to correct folding. The experimental conditions for refolding may play a role in defining the function of GroEL in the folding pathway. We propose that the role of GroES and MgATP, either binding or hydrolysis, is to regulate the association and dissociation processes rather than affecting the rate of folding.
Mendel’s dwarfing gene: cDNAs from the
Le
alleles and function of the expressed proteins
Authors: Martin David N. , Proebsting William M. , Hedden Peter
Published: July 2002

The major gibberellin (GA) controlling stem elongation in pea (
Pisum sativum
L.) is GA
1
, which is formed from GA
20
by 3β-hydroxylation. This step, which limits GA
1
biosynthesis in pea, is controlled by the
Le
locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3β-hydroxylase from lines of pea carrying the
Le
,
le
,
le-3
, and
le
d
alleles. The cDNA sequences from
le
and
le-3
each contain a base substitution resulting in single amino acid changes relative to the sequence from
Le
. The cDNA sequence from
le
d
, a mutant derived from an
le
line, contains both the
le
“mutation” and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in
Escherichia coli
. The expression product for the clone from
Le
converted GA
9
to GA
4
, and GA
20
to GA
1
, with K
m
values of 1.5 μM and 13 μM, respectively. The amino acid substitution in the clone from
le
increased K
m
for GA
9
100-fold and reduced conversion of GA
20
to almost nil. Expression products from
le
and
le-3
possessed similar levels of 3β-hydroxylase activity, and the expression product from
le
d
was inactive. Our results suggest that the 3β-hydroxylase cDNA is encoded by
Le. Le
transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.
Cancer-associated mutations at the
INK4a
locus cancel cell cycle arrest by p16
INK4a
but not by the alternative reading frame protein p19
ARF
Authors: Quelle Dawn E. , Cheng Mangeng , Ashmun Richard A. , Sherr Charles J.
Published: July 2002

The
INK4a
gene, one of the most frequently disrupted tumor suppressor loci in human cancer, encodes two unrelated proteins, p16
INK4a
and p19
ARF
, each of which is capable of inducing cell cycle arrest. Splicing of alternative first exons (1α vs. 1β) to a common second exon within
INK4a
generates mRNAs in which exon 2 sequences are translated in two different reading frames. One of the products, the cyclin D-dependent kinase inhibitor p16
INK4a
, is functionally inactivated by mutations or deletions in a wide variety of cancers. However, because many such mutations reside in exon 2, they also affect the alternative reading frame (ARF) protein. To determine whether such mutations disrupt p19
ARF
function, we introduced naturally occurring missense mutations into mouse
INK4a
exon 2 sequences and tested mutant p16
INK4a
and p19
ARF
proteins for their ability to inhibit cell cycle progression. Six p19
ARF
point mutants remained fully active in mediating cell cycle arrest in NIH 3T3 fibroblasts, whereas two of the corresponding mutations within p16
INK4a
resulted in complete loss of activity. Analysis of p19
ARF
deletion mutants indicated that the unique aminoterminal domain encoded by exon 1β was both necessary and sufficient for inducing G
1
arrest. Therefore, cancer-associated mutations within exon 2 of the
INK4a
gene specifically target p16
INK4a
, and not p19
ARF
, for inactivation.
Methylation of the minimal promoter of an embryonic globin gene silences transcription in primary erythroid cells
Authors: Singal Rakesh , Ferris Richard , Little Jane A. , Wang Shu Zhen , Ginder Gordon D.
Published: July 2002

Methylation of cytosines in the dinucleotide CpG has been shown to suppress transcription of a number of tissue-specific genes, yet the precise mechanism is not fully understood. The vertebrate globin genes were among the first examples in which an inverse correlation was shown between CpG methylation and transcription. We studied the methylation pattern of the 235-bp ρ-globin gene promoter in genomic DNA from primary chicken erythroid cells using the sodium bisulfite conversion technique and found all CpGs in the promoter to be methylated in erythroid cells from adult chickens in which the ρ-globin gene is silent but unmethylated in 5-day (primitive) embryonic red cells in which the gene is transcribed. To elucidate further the mechanism of methylation-induced silencing, an expression construct consisting of 235 bp of 5′ promoter sequence of the ρ-globin gene along with a strong 5′ erythroid enhancer driving a chloramphenicol acetyltransferase reporter gene, ρ-CAT, was transfected into primary avian erythroid cells derived from 5-day embryos. Methylation of just the 235-bp ρ-globin gene promoter fragment at every CpG resulted in a 20- to 30-fold inhibition of transcription, and this effect was not overridden by the presence of potent erythroid-specific enhancers. The ability of the 235-bp ρ-globin gene promoter to bind to a DNA
Me
thyl
C
ytosine binding
P
rotein
C
omplex (MeCPC) was tested in electrophoretic mobility shift assays utilizing primary avian erythroid cell nuclear extract. The results were that fully methylated but not unmethylated 235-bp ρ-globin gene promoter fragment could compete efficiently for MeCPC binding. These results are a direct demonstration that site-specific methylation of a globin gene promoter at the exact CpGs that are methylated
in vivo
can silence transcription in homologous primary erythroid cells. Further, these data implicate binding of MeCPC to the promoter in the mechanism of silencing.
Interferon-γ impacts at multiple points during the progression of autoimmune diabetes
Authors: Wang Bo , André Isabelle , Gonzalez Antonio , Katz Jonathan D. , Aguet Michel , Benoist Christophe , Mathis Diane
Published: July 2002

The role of interferon-γ in autoimmune diabetes was assessed by breeding a null mutation of the interferon-γ receptor α chain into the nonobese diabetic mouse strain, as well as into a simplified T cell receptor transgenic model of diabetes. In contrast to a previous report on abrogation of the interferon-γ gene, mutation of the gene encoding its receptor led to drastic effects on disease in both mouse lines. Nonobese diabetic mice showed a marked inhibition of insulitis—both the kinetics and penetrance—and no signs of diabetes; the transgenic model exhibited near-normal insulitis, but this never evolved into diabetes, either spontaneously or after experimental provocation. This failure could not be explained by perturbations in the ratio of T helper cell phenotypes; rather, it reflected a defect in antigen-presenting cells or in the islet β cell targets.
Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS–Kaposi sarcoma
Authors: Masood Rizwan , Cai Jie , Zheng Tong , Smith D. Lynne , Naidu Yathi , Gill Parkash S.
Published: July 2002

Kaposi sarcoma (KS) is the most common tumor associated with HIV-1 infection and develops in nearly 30% of cases. The principal features of this tumor are abnormal vascularization and the proliferation of endothelial cells and spindle (tumor) cells. KS-derived spindle cells induce vascular lesions and display enhanced vascular permeability when inoculated subcutaneously in the nude mouse. This finding suggests that angiogenesis and capillary permeability play a central role in the development and progression of KS. In this study, we show that AIDS–KS cell lines express higher levels of vascular endothelial growth factor/vascular permeability factor (VEGF/VGF) than either human umbilical vein endothelial cells or human aortic smooth muscle cells. AIDS–KS cells and primary tumor tissues also expressed high levels of Flt-1 and KDR, the receptors for VEGF, while the normal skin of the same patients did not show any expression. We further demonstrate that VEGF antisense oligonucleotides AS-1 and AS-3 specifically block VEGF mRNA and protein production and inhibit KS cell growth in a dose-dependent manner. Furthermore, growth of KS cells in nude mice was specifically inhibited by VEGF antisense oligonucleotides. These results show that VEGF is an autocrine growth factor for AIDS–KS cells. To our knowledge, this is the first report that shows that VEGF acts as a growth stimulator in a human tumor. Inhibitors of VEGF or its cognate receptors may thus be candidates for therapeutic intervention.
Genetically engineered superantigens as tolerable antitumor agents
Authors: Hansson Johan , Ohlsson Lennart , Persson Robert , Andersson Gunnar , Ilbäck Nils-Gunnar , Litton Mark J. , Kalland Terje , Dohlsten Mikael
Published: July 2002

Superantigens (SAg) are a family of bacterial and viral proteins with strong immunostimulatory properties. SAg bound to major histocompatibility complex (MHC) class II molecules activate a high frequency of T cells and represent the most potent known activators of T cells to date. To explore the use of SAg for T cell-based tumor therapy we have created a tumor-reactive SAg by engineering a fusion protein composed of a tumor-reactive mAb (C215Fab) and the bacterial SAg staphylococcal enterotoxin A (SEA). A point mutation D227A was introduced at the major MHC class II binding site in SEA to reduce systemic toxicity. Treatment of tumor bearing mice with the Fab–SEA D227A fusion protein resulted in profound antitumor effects with a markedly reduced toxicity as compared with the wild-type Fab–SEA fusion protein. The reduced toxicity was probably due to a weak distribution of the SEA D227A fusion protein in tissues with a high MHC class II expression and low systemic cytokine levels as exhibited in mice and rabbits. The data presented demonstrate the efficacy of immunoconjugates containing a mutated SAg in directing a T cell attack against tumor cells with minimal systemic immune activation.
A genetic system for Archaea of the genus
Methanosarcina
: Liposome-mediated transformation and construction of shuttle vectors
Authors: Metcalf William W. , Zhang Jun Kai , Apolinario Ethel , Sowers Kevin R. , Wolfe Ralph S.
Published: July 2002

New methods that allow, for the first time, genetic analysis in Archaea of the genus
Methanosarcina
are presented. First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from
Methanosarcina acetivorans
. These vectors replicate in 9 of 11
Methanosarcina
strains tested and in
Escherichia coli
. Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed. This method allows transformation frequencies of as high as 2 × 10
8
transformants per microgram of DNA per 10
9
cells or ≈20% of the recipient population. During the course of this work, the complete 5467-bp DNA sequence of pC2A was determined. The implications of these findings for the future of methanoarchaeal research are also discussed.