bluuebunny/crossref_metadata_2025 huggingface dataset:
An Essential Role ofS-Adenosyl-l-Methionine:l-MethionineS-Methyltransferase in Selenium Volatilization by Plants. Methylation of Selenomethionine to Selenium-Methyl-l-Selenium- Methionine, the Precursor of Volatile Selenium
Authors: Tagmount Abderrahmane , Berken Antje , Terry Norman
Published: October 2002

Selenium (Se) phytovolatilization, the process by which plants metabolize various inorganic or organic species of Se (e.g. selenate, selenite, and Se-methionine [Met]) into gaseous Se forms (e.g. dimethylselenide), is a potentially important means of removing Se from contaminated environments. Before attempting to genetically enhance the efficiency of Se phytovolatilization, it is essential to elucidate the enzymatic pathway involved and to identify its rate-limiting steps. The present research tested the hypothesis thatS-adenosyl-l-Met:l-MetS-methyltransferase (MMT) is the enzyme responsible for the methylation of Se-Met to Se-methyl Se-Met (SeMM). To this end, we identified and characterized an Arabidopsis T-DNA mutant knockout for MMT. The lack of MMT in the Arabidopsis T-DNA mutant plant resulted in an almost complete loss in its capacity for Se volatilization. Using chemical complementation with SeMM, the presumed enzymatic product of MMT, we restored the capacity of the MMT mutant to produce volatile Se. Overexpressing MMT from Arabidopsis in Escherichia coli, which is not known to have MMT activity, produced up to 10 times more volatile Se than the untransformed strain when both were supplied with Se-Met. Thus, our results provide in vivo evidence that MMT is the key enzyme catalyzing the methylation of Se-Met to SeMM.
Characterization of SP1, a Stress-Responsive, Boiling-Soluble, Homo-Oligomeric Protein from Aspen
Authors: Wang Wang-Xia , Pelah Dan , Alergand Tal , Shoseyov Oded , Altman Arie
Published: October 2002

sp1 cDNA was isolated from aspen (Populus tremula) plants by immunoscreening an expression library using polyclonal antibodies against BspA protein. BspA, which is a boiling-stable protein, accumulates in aspen plants in response to water stress and abscisic acid application (Pelah et al., 1995). The sp1 cDNA was found to encode a 12.4-kD generally hydrophilic protein with a hydrophobic C terminus, which is different from the BspA protein and was termed SP1 (stable protein 1). Northern-blot analysis revealed that sp1 encodes a small mRNA (about 0.6 kb) that is expressed in aspen plants under non-stress conditions and is accumulated after salt, cold, heat, and desiccation stress, and during the recovery from stress. The SP1 detected in plants remained soluble upon boiling, migrated both as a 12.4-kD band and a much higher mass of 116 kD on a 17% (w/v) Tricine-sodium dodecyl sulfate-polyacrylamide gel. Comparative protease digestion patterns, amino acid analyses, and the N-terminal sequences of the 12.4- and 116-kD proteins revealed that SP1 is homo-oligomeric. Furthermore, gel filtration chromatography analysis indicated that SP1 exists in aspen plants as a complex, composed of 12 subunits of 12.4 kD. A large number of sequences deduced from expressed sequence tags and genomic sequences of other organisms with unknown function show high homology to SP1. Thus, SP1 may represent a new protein family. Here, we present the first report on this putative protein family: the cloning, isolation, and characterization of SP1, a stress-responsive, boiling-soluble, oligomeric protein.
Hydrogen Peroxide Activates Cell Death and Defense Gene Expression in Birch
Authors: Pellinen Riikka I. , Korhonen Minna-Sisko , Tauriainen Airi A. , Palva E. Tapio , Kangasjärvi Jaakko
Published: October 2002

The function of hydrogen peroxide (H2O2) as a signal molecule regulating gene expression and cell death induced by external stresses was studied in birch (Betula pendula). Ozone (O3),Pseudomonas syringae pv syringae(Pss), and wounding all induced cell death of various extents in birch leaves. This was temporally preceded and closely accompanied by H2O2 accumulation at, and especially surrounding, the lesion sites. O3 andPss, along with an artificial H2O2 producing system glucose (Glc)/Glc oxidase, elicited elevated mRNA levels corresponding to genes encoding reactive oxygen species detoxifying enzymes, Pal,Ypr10, and mitochondrial phosphate translocator 1. In addition to the regulation of gene expression, Glc/Glc oxidase also induced endogenous H2O2 production in birch leaves, accompanied by cell death that resembled O3 andPss damage. Wound-induced gene expression differed from that induced by O3 and Pss. Thus, it appears that at least two separate defense pathways can be activated in birch leaves by stress factors, even though the early H2O2 accumulation response is common among them all.
An Arabidopsis Mutant Defective in Jasmonate Response Is Allelic to the Auxin-Signaling Mutant axr1
Authors: Tiryaki Iskender , Staswick Paul E.
Published: October 2002

A screen for Arabidopsis mutants that were insensitive to methyl jasmonate (MeJA) in an assay for seedling root growth yielded only alleles of previously isolated mutants jar1 andcoi1, with one exception. Mapping of the locus and morphological characterization of the new mutant suggested it might be allelic to axr1, which had not previously been reported to show resistance to MeJA. The F1 from a cross of the new mutant with axr1-3 did not show complementation, confirming that these are the same genes. The new allele is calledaxr1-24. In addition to MeJA and indole-3-acetic acid (IAA), axr1-24 had decreased sensitivity to 1-aminocyclopropane-1-carboxylic acid, 6-benzylamino-purine, epi-brassinolide, and abscisic acid. Both axr1-24 and the previously characterized axr1-3 allele were shown to be susceptible to the opportunistic pathogen Pythium irregulare, a trait found in other jasmonate response mutants, including jar1-1. The double mutantjar1-1/axr1-3 was more resistant to inhibition of root growth by MeJA and was more susceptible to P. irregulare infection than either single mutant, suggesting these genes might act in independent response pathways. In contrast, resistance to IAA in the double mutant was not different fromaxr1-3. Northern-blot analysis showed that IAA induced the jasmonate-responsive lipoxygenase 2,AOS, and AtVSP gene transcripts and induction was strongly impaired in axr1-3. However, transcript induction by MeJA was only minimally affected inaxr1-3. This study demonstrates that in addition to auxin signaling, the AXR1 locus is involved in MeJA response, providing a mechanistic link between jasmonate and auxin-signaling pathways.
Identification and Biochemical Characterization of Mutants in the Proanthocyanidin Pathway in Arabidopsis
Authors: Abrahams Sharon , Tanner Gregory J. , Larkin Philip J. , Ashton Anthony R.
Published: October 2002

Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO4) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds(tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3,tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutantstds1, tds2, tds3, andtds5 (which do not produce PA) and tds6(6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 andtt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation.
Transcription Profiling of the Early Gravitropic Response in Arabidopsis Using High-Density Oligonucleotide Probe Microarrays,
Authors: Moseyko Nick , Zhu Tong , Chang Hur-Song , Wang Xun , Feldman Lewis J.
Published: October 2002

Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.
Separable Roles for rent1/hUpf1 in Altered Splicing and Decay of Nonsense Transcripts
Authors: Mendell Joshua T. , ap Rhys Colette M. J. , Dietz Harry C.
Published: October 2002

The mechanism by which disruption of reading frame can influence pre–messenger RNA (pre-mRNA) processing is poorly understood. We assessed the role of factors essential for nonsense-mediated mRNA decay (NMD) in nonsense-mediated altered splicing (NAS) with the use of RNA interference (RNAi) in mammalian cells. Inhibition of rent1/hUpf1 expression abrogated both NMD and NAS of nonsense T cell receptor β transcripts. In contrast, inhibition of rent2/hUpf2 expression did not disrupt NAS despite achieving comparable stabilization of nonsense transcripts. We also demonstrate that NAS and NMD are genetically separable functions of rent1/hUpf1. Additionally, rent1/hUpf1 enters the nucleus where it may directly influence early events in mRNA biogenesis. This provides compelling evidence that NAS relies on a component of the nonsense surveillance machinery but is not an indirect consequence of NMD.
Nanometer Resolution and Coherent Optical Dipole Coupling of Two Individual Molecules
Authors: Hettich C. , Schmitt C. , Zitzmann J. , Kühn S. , Gerhardt I. , Sandoghdar V.
Published: October 2002

By performing cryogenic laser spectroscopy under a scanning probe electrode that induces a local electric field, we have resolved two individual fluorescent molecules separated by 12 nanometers in an organic crystal. The two molecules undergo a strong coherent dipole-dipole coupling that produces entangled sub- and superradiant states. Under intense laser illumination, both molecules are excited via a two-photon transition, and the fluorescence from this doubly excited system displays photon bunching. Our experimental scheme can be used to optically resolve molecules at the nanometer scale and to manipulate the degree of entanglement among them.
A Classical Nova, V2487 Oph 1998, Seen in X-rays Before and After Its Explosion
Authors: Hernanz Margarita , Sala Glòria
Published: October 2002

Classical nova explosions are very energetic and frequent phenomena caused by explosive hydrogen burning on top of an accreting white dwarf. Observations of the recent nova V2487 Oph 1998 by the X-ray Multi-Mirror satellite (XMM-Newton) provide evidence that accretion (probably on a magnetic white dwarf) was reestablished as early as 2.7 years after the explosion. In addition, positional correlation with a source previously discovered by the Röntgen Satellite (ROSAT) in 1990 suggests that the site of a nova explosion had been seen in x-rays before the outburst.
Potential for using antibiotics combined with a Shiga toxin-absorbing agent for treating 0157:H7 Escherichia coli infections
Authors: Mulvey George , Rafter David J , Armstrong Glen D
Published: October 2002

Antibiotics are not recommended for treating O157:H7 Escherichia coli infections because they may promote Shiga toxin (Stx) release from these bacteria. This could increase the risk of Stx-mediated complications in patients suffering from such infections. Here, we observed increased cell-free Stx in E. coli O157:H7 cultures exposed to sub-inhibitory concentrations of several antibiotics. Synsorb-Pk, an agent with a high affinity for Stx, absorbed Stx activity from the antibiotic-treated cultures. These data suggest certain antibiotics, given in combination with an orally administered Stx-binding agent, may be useful in treating O157:H7 E. coli infections.Key words: Shiga toxin, Synsorb, Escherichia coli, O157:H7, antibiotics, therapy.
Examination of the structural basis for O(H) blood group specificity byUlex europaeusLectin I
Authors: Audette Gerald F , Olson Douglas J , Ross Andrew R , Quail J Wilson , Delbaere Louis T
Published: October 2002

The structural basis for carbohydrate specificity of the first lectin from Ulex europaeus (UE-I) is reported. UE-I is a dimeric metalloglycoprotein that binds the H-type 2 human blood group determinant (α-L-Fucα(1[Formula: see text]2)-β;-D-Galβ(1[Formula: see text]4)-β-D-GlcNAcα-), the blood group determinant present on the surface of O-type erythrocytes. The structural characteristics of UE-I involved in carbohydrate recognition have been examined using mass spectrometry (MS) and X-ray diffraction analysis. MS analysis allowed for discrimination between the different primary structures reported for UE-I. To examine the binding of the H-type 2 blood group determinant by UE-I, the methyl glycosides of the fucose monosaccharide (α-L-Fuc-OMe), known to exhibit primary binding specificity, and the H-type 2 trisaccharide (H-type 2-OMe) were, in two separate experiments, co-crystallized into the binding site of UE-I. The UE-I:α-L-Fuc-OMe complex crystallizes in the monoclinic space group P21, with unit cell dimensions a = 71.81, b = 69.00, and c = 119.02 Å, and β = 106.76°. Two UE-I dimers are observed to be present within the asymmetric unit, and the model has been refined to a R-value and RFreeof 0.202 and 0.289, respectively, to 2.3 Å resolution. The preliminary model of the UE-I:H-type 2-OMe complex has been refined at 3.0 Å resolution. The UE-I:H-type 2-OMe complex crystallizes in the orthorhombic space group C2221, with unit cell dimensions a = 88.80, b = 164.75, and c = 77.42 Å, and a single UE-I dimer is present within the asymmetric unit. The carbohydrate recognition domain of UE-I has been identified to be comprised of residues Glu44, Thr86, Asp87, Arg102, Ala103, Gly104, Gly105, Tyr106, Ile129, Val133, Asn134, Trp136, Tyr219, and Arg222. Several critical protein-carbohydrate interactions have been identified, including the role of the hydrophobic interaction between the Thr86 side chain and C-5-CH3of the α-L-Fuc-OMe. The role of these interactions in carbohydrate recognition-binding by UE-I, as well as differences between the observed and previously modeled complexes, are discussed. Key words: Ulex europaeus lectin I, H-type 2 human blood group determinant, protein-carbohydrate interactions, X-ray crystallography, chemical mapping.
Modeling in-situ pine root decomposition using data from a 60-year chronosequence
Authors: Ludovici Kim H , Zarnoch Stanley J , Richter Daniel D
Published: October 2002

Because the root system of a mature pine tree typically accounts for 2030% of the total tree biomass, decomposition of large lateral roots and taproots following forest harvest and re-establishment potentially impact nutrient supply and carbon sequestration in pine systems over several decades. If the relationship between stump diameter and decomposition of taproot and lateral root material, i.e., wood and bark, can be quantified, a better understanding of rates and patterns of sequestration and nutrient release can also be developed. This study estimated decomposition rates from in-situ root systems using a chronosequence approach. Nine stands of 55- to 70-year-old loblolly pine (Pinus taeda L.) that had been clear-cut 0, 5, 10, 20, 25, 35, 45, 55, and 60 years ago were identified on well-drained Piedmont soils. Taproot and lateral root systems were excavated, measured, and weighed. Although more than 50% of the total root mass decomposed during the first 10 years after harvest, field excavations recovered portions of large lateral roots (>5 cm diameter) and taproots that persisted for more than 35 and 60 years, respectively. Results indicate that decomposition of total root biomass, and its component parts, from mature, clear-cut loblolly pine stands, can be modeled with good precision as a function of groundline stump diameter and years since harvest.
Development of Norway spruce dominated stands after single-tree selection and low thinning
Authors: Lähde Erkki , Laiho Olavi , Norokorpi Yrjö , Saksa Timo
Published: October 2002

The study included 23 stands (at least 2 ha each in size) distributed from southern to northern Finland. These Norway spruce (Picea abies (L.) Karst.) dominated stands grew on fertile (OxalisMyrtillus and Myrtillus site types) mineral soils. Each stand contained two substands randomly treated with single-tree selection or low thinning. The harvested volumes (trees > 9 cm) varied greatly but averaged 94 m3·ha1 in the former consisting of mainly medium-sized and larger (>15 cm) trees and 68 m3·ha1 in the latter of mainly medium-sized and smaller (<15 cm) trees. After treatment, mean diameter at breast height (DBH), basal area, and stand volume were 1217% lower in single-tree selection than in low thinning. The stem distributions were reverse-J shaped and bell shaped, respectively. During the monitoring of a mean of 11 years, about one cutting cycle in single-tree selection, stand volume (trees > 9 cm) increased 38% in single-tree selection and 27% in low thinning. The respective current annual volume and relative increments were 5.4 (3.6%) and 4.6 m3·ha1·year1 (2.4%). In 18 (volume) and in 22 (relative) of the 23 plot pairs the increment was higher after single-tree selection than after low thinning (p values 0.013 and <0.001, respectively). Single-tree selection plots additionally included 1300 saplings/ha (from breast height to DBH 9 cm) after cutting, with the transition of 80 saplings/ha into larger trees and with the ingrowth, mainly spruce, of 170 seedlings/ha into saplings during monitoring.
Grizzly bear use of open, closed, and restricted forestry roads
Authors: Wielgus Robert B , Vernier Pierre R , Schivatcheva Tina
Published: October 2002

We investigated grizzly bear (Ursus arctos) selection of three road types in the northern United States and southern British Columbia from 1986 to 1991. We hypothesized that grizzly bears select against open (public use allowed), restricted (forestry use only), and closed roads (no public use allowed) in that order. We analyzed use of roads for 11 bears (five females and six males) in an area containing open and closed roads and 11 bears (seven females and four males) in an adjacent area containing restricted roads. We used χ2 and loglinear models to test for selection of habitat type and distance to road categories. Ten of 12 females and 5 of 10 males (15 of 22 bears) selected against (P < 0.05) low-elevation interior cedar-hemlock and for (P < 0.05) high-elevation Englemann spruce (Picea engelmannii Parry ex Engelm.)  subalpine fir (Abies lasiocarpa (Hook.) Nutt.). After accounting for habitat, 4 of 5 females and 3 of 6 males (7 of 11 bears) selected against open roads and 3 of 5 females and 0 of 6 males (3 of 11 bears) selected against closed roads. No females (n = 7) or males (n = 4) (0 of 11 bears) selected against restricted roads. Our results are inconsistent with the hypothesis that bears select against open, restricted, and closed roads in that order. Most females and males selected against open roads, most females selected against closed roads, and no bears selected against restricted roads. The type of human activity along roads plays a role in bear responses to roads, and this aspect should be incorporated into future bear-road studies.
MICROCANONICAL DETERMINATION OF THE ORDER PARAMETER CRITICAL EXPONENT
Authors: HÜLLER ALFRED , PLEIMLING MICHEL
Published: October 2002

A highly efficient Monte Carlo method for the calculation of the density of states of classical spin systems is presented. As an application, we investigate the density of states ΩN(E, M) of two- and three-dimensional Ising models with N spins as a function of energy E and magnetization M. For a fixed energy lower than a critical value Ec,N the density of states exhibits two sharp maxima at M = ± Msp(E) which define the microcanonical spontaneous magnetization. An analysis of the form Msp(E) ∝ (Ec, ∞ - E)βε yields very good results for the critical exponent βε, thus demonstrating that critical exponents can be determined by analyzing directly the density of states of finite systems.
POSSIBLE ASTROPHYSICAL PROBES OF QUANTUM GRAVITY
Authors: SARKAR SUBIR
Published: October 2002

A satisfactory theory of quantum gravity will very likely require modification of our classical perception of space-time, perhaps by giving it a 'foamy' structure at scales of order the Planck length. This is expected to modify the propagation of photons and other relativistic particles such as neutrinos, such that they will experience a non-trivial refractive index even in vacuo. The implied spontaneous violation of Lorentz invariance may also result in alterations of kinematical thresholds for key astrophysical processes involving high energy cosmic radiation. We discuss experimental probes of these possible manifestations of the fundamental quantum nature of space-time using observations of distant astrophysical sources such as gamma-ray bursts and active galactic nuclei.
REFLECTIONS ON THE SPATIOTEMPORAL ASPECTS OF THE QUANTUM WORLD
Authors: MOHRHOFF ULRICH
Published: October 2002

The proper resolution of the so-called measurement problem requires a "top-down" conception of the quantum world that is opposed to the usual "bottom-up" conception, which builds on an intrinsically and maximally differentiated manifold. The key to that problem is that the fuzziness of a variable can manifest itself only to the extent that less fuzzy variables exist. Inasmuch as there is nothing less fuzzy than the metric, this argues against a quantum-gravity phenomenology and suggests that a quantum theory of gravity is something of a contradiction in terms — a theory that would make it possible to investigate the physics on scales that do not exist, or to study the physical consequences of a fuzziness that has no physical consequences, other than providing a natural cutoff for the quantum field theories of particle physics.
Many-Layered Learning
Authors: Utgoff Paul E. , Stracuzzi David J.
Published: October 2002

We explore incremental assimilation of new knowledge by sequential learning. Of particular interest is how a network of many knowledge layers can be constructed in an on-line manner, such that the learned units represent building blocks of knowledge that serve to compress the overall representation and facilitate transfer. We motivate the need for many layers of knowledge, and we advocate sequential learning as an avenue for promoting the construction of layered knowledge structures. Finally, our novel STL algorithm demonstrates a method for simultaneously acquiring and organizing a collection of concepts and functions as a network from a stream of unstructured information.
Mineralogy of complex Co-Ni-Bi vein mineralization, Bieber deposit, Spessart, Germany
Authors: Wagner T. , Lorenz J.
Published: October 2002

Post-Variscan vein-type Co-Ni-Bi ores of the Bieber deposit, Spessart mountains, Germany, which are related to the Permian Kupferschiefer, have been investigated by ore microscopy, X-ray powder diffraction and electron-probe microanalysis. The samples contain a variety of ore minerals, notably skutterudite, native bismuth, cobaltite, alloclasite, niccolite, maucherite, gersdorffite, rammelsbergite/pararammelsbergite, safflorite, loellingite and emplectite. The ores display structures indicative of multiple brecciation and complex zoned arsenide assemblages. Three sequential stages of deposition are identified, which are (1) the Cu stage, (2) the main Co-Ni-Bi stage, and (3) the late stage. The arsenide minerals, notably skutterudite, diarsenides and sulpharsenides, show a large range of compositional variation in Co-Ni-Fe space. A relatively limited number of skutterudite and diarsenide compositions lie outside the compositional fields established in the literature. Skutterudite and diarsenides are characterized by a significant substitution of As by S up to 0.44 a.p.f.u. and 0.31 a.p.f.u., respectively, which is larger than the range previously reported for these minerals. Sulpharsenide compositions can be grouped into three populations, which conform to cobaltian arsenopyrite, cobaltite and gersdorffite. They display highly variable As/S ratios between 0.95:1.00 and 1.29:0.73, consistent with experimental data. Estimates of the formation temperatures, based on the presence of dendritic native bismuth and emplectite, are in the range 100–300°C, similar to different post-Variscan mineralization styles widespread in Central Europe. Comparison of the Co-Ni-Bi vein assemblage with the framework of available paragenetic information and radiometric age data for regional mineralization events indicates an age of mineralization of ~150–160 Ma for the Bieber deposit.
Short deletion within the blood group Dombrock locus causing a Donull phenotype
Authors: Lucien Nicole , Celton Jean-Louis , Pennec Pierre-Yves Le , Cartron Jean-Pierre , Bailly Pascal
Published: October 2002

A new alteration of the blood group DO*A allele was identified in a female Donull donor from Reunion Island with allo– anti-DO3 in her serum; her parents are consanguineous. Because the amplification of the DO transcript failed, each exon and intron–exon junction from the DO gene were examined. After polymerase chain reaction (PCR) amplification and sequencing, the only deviation from the wild-type DO*A allele sequence was an 8-nucleotide deletion (nt 343-350) within exon 2. This short deletion generates a premature stop codon and encodes a truncated protein lacking the predicted functional motif of the adenosine diphosphate–ribosyltransferase enzyme and the glycosyl-phosphatidylinositol anchor motif essential for RBC membrane attachment. An allele-specific PCR to detect the DO(Δ8nt) deletion was developed.
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) acts as a regulator of B-cell development, B-cell antigen receptor (BCR)–mediated activation, and autoimmune disease
Authors: Wilkinson Ray , Lyons A. Bruce , Roberts Donna , Wong Mae-Xhum , Bartley Paul A. , Jackson Denise E.
Published: October 2002

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is an immunoglobulin–immunoreceptor tyrosine-based inhibitory motif (Ig-ITIM) superfamily member that recruits and activates protein-tyrosine phosphatases, SHP-1 and SHP-2, through its intrinsic ITIMs. PECAM-1–deficient (PECAM-1−/− ) mice exhibit a hyperresponsive B-cell phenotype, increased numbers of B-1 cells, reduced B-2 cells, and develop autoantibodies. In the periphery, there are reduced mature recirculating B-2 cells and increased B-1a cells within the peritoneal cavity. In addition, PECAM-1−/− B cells display hyperproliferative responses to lipopolysaccharide and anti-IgM stimulation and showed enhanced kinetics in their intracellular Ca++ response following IgM cross-linking. PECAM-1−/− mice showed increased serum levels of IgM with elevated IgG isotypes and IgA antidinitrophenol antibody in response to the T-independent antigen, dinitrophenol-Ficoll. Finally, PECAM-1−/− mice developed antinuclear antibodies and lupuslike autoimmune disease with age.
Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development
Authors: Hanspal Manjit , Dua Meenakshi , Takakuwa Yuichi , Chishti Athar H. , Mizuno Akiko
Published: October 2002

Plasmodium falciparum–derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2–mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin–actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2–induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo.
Allogeneic bone marrow transplantation: cure for familial Mediterranean fever
Authors: Milledge John , Shaw Peter J. , Mansour Albert , Williamson Sarah , Bennetts Bruce , Roscioli Tony , Curtin Julie , Christodoulou John
Published: October 2002

We describe data on a 7-year-old girl with congenital dyserythropoietic anemia (CDA), who also had familial Mediterranean fever (FMF). Repeated transfusions required since the age of 6 months to treat her CDA led to iron overload and a persistently high ferritin level. Her relapsing FMF made effective iron chelation therapy very difficult. Consequently, at the age of 4 years, she underwent allogeneic, sibling bone marrow transplantation (BMT). During conditioning for her BMT, symptoms of FMF, including splenomegaly, arthritis, and recurrent abdominal pain, began to resolve and she was gradually weaned off colchicine. Now, 2 years after the transplantation, she remains free from FMF symptomatology and is off all immunosuppressants. This case demonstrates that symptoms of FMF can be alleviated by the therapy used during allogeneic BMT. In this patient it is likely that the missing factor in FMF is now being provided by granulocytes derived from the stem cells within transplanted bone marrow.
Functional analysis of human hematopoietic repopulating cells mobilized with granulocyte colony-stimulating factor alone versus granulocyte colony-stimulating factor in combination with stem cell factor
Authors: Hess David A. , Levac Krysta D. , Karanu Francis N. , Rosu-Myles Michael , White Martin J. , Gallacher Lisa , Murdoch Barbara , Keeney Michael , Ottowski Pamela , Foley Ronan , Chin-Yee Ian , Bhatia Mickie
Published: October 2002

Using in vitro progenitor assays, serum-free in vitro cultures, and the nonobese diabetic/severe combined immune-deficient (NOD/SCID) ecotropic murine virus knockout xenotransplantation model to detect human SCID repopulating cells (SRCs) with multilineage reconstituting function, we have characterized and compared purified subpopulations harvested from the peripheral blood (PB) of patients receiving granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF). Mobilized G-CSF plus SCF PB showed a 2-fold increase in total mononuclear cell content and a 5-fold increase in CD34-expressing cells depleted for lineage-marker expression (CD34+Lin−) as compared with patients treated with G-CSF alone. Functionally, G-CSF plus SCF–mobilized CD34+CD38−Lin−cells contained a 2-fold enhancement in progenitor frequency as compared with G-CSF–mobilized subsets. Despite enhanced cellularity and progenitor capacity, G-CSF plus SCF mobilization did not increase the frequency of SRCs as determined by limiting dilution analysis by means of unfractionated PB cells. Purification of SRCs from these sources demonstrated that as few as 1000 CD34+CD38−Lin− cells from G-CSF–mobilized PB contained SRC capacity while G-CSF plus SCF–mobilized CD34+CD38−Lin−cells failed to repopulate at doses up to 500 000 cells. In addition, primitive CD34−CD38−AC133+Lin−cells derived from G-CSF plus SCF–mobilized PB were capable of differentiation into CD34-expressing cells, while the identical subfractions from G-CSF PB were unable to produce CD34+cells in serum-free cultures. Our study defines qualitative and quantitative distinctions among subsets of primitive cells mobilized by means of G-CSF plus SCF versus G-CSF alone, and therefore has implications for the utility of purified repopulating cells from these sources.
Human cytomegalovirus inhibits maturation and impairs function of monocyte-derived dendritic cells
Authors: Moutaftsi Magdalena , Mehl Anja M. , Borysiewicz Leszek K. , Tabi Zsuzsanna
Published: October 2002

Dendritic cells (DCs) play a pivotal role in the generation of virus-specific cytotoxic T-cell responses, but some viruses can render DCs inefficient in stimulating T cells. We studied whether infection of DCs with human cytomegalovirus (HCMV) results in a suppression of DC function which may assist HCMV in establishing persistence. The effect of HCMV infection on the phenotype and function of monocyte-derived DCs and on their ability to mature following infection with an endothelial cell–adapted clinical HCMV isolate were studied. HCMV infection induced no maturation of DCs; instead, it efficiently down-regulated the expression of surface major histocompatibility complex (MHC) class I, CD40, and CD80 molecules. Slight down-regulation of MHC class II and CD86 molecules was also observed. Lipopolysaccharide (LPS)–induced maturation of infected DCs was strongly inhibited, as indicated by lower levels of surface expression of MHC class I, class II, costimulatory, and CD83 molecules. The down-regulation or inhibition of these surface markers occurred only in HCMV antigen-positive DCs. DCs produced no interleukin 12 (IL-12) and only low levels of tumor necrosis factor alpha (TNF-α) upon HCMV infection. Furthermore, cytokine production upon stimulation with LPS or CD40L was significantly impaired. Inhibition of cytokine production did not depend on viral gene expression as UV-irradiated HCMV resulted in the same effect. Proliferation and cytotoxicity of T cells specific to a recall antigen presented by DCs were also reduced when DCs were HCMV infected. This study shows that HCMV inhibits DC function, revealing a powerful viral strategy to delay or prevent the generation of virus-specific cytotoxic T cells.
Presence of a mobilizable intracellular pool of hepatocyte growth factor in human polymorphonuclear neutrophils
Authors: Grenier Alain , Chollet-Martin Sylvie , Crestani Bruno , Delarche Charlotte , El Benna Jamel , Boutten Anne , Andrieu Valérie , Durand Geneviève , Gougerot-Pocidalo Marie-Anne , Aubier Michel , Dehoux Monique
Published: October 2002

Hepatocyte growth factor (HGF), a heparin-binding factor, is synthesized as a single-chain inactive precursor (pro-HGF), which is converted by proteolysis to an active heterodimer (mature HGF). HGF has pleiotropic activities and has been implicated in the regulation of mitogenesis, motogenesis, and morphogenesis of epithelial and endothelial cells. As polymorphonuclear neutrophils (PMNs) secrete numerous cytokines involved in the modulation of local inflammation, we investigated their ability to produce HGF. We found that HGF was stored in secretory vesicles and in gelatinase/specific granules. This intracellular stock was rapidly mobilized by degranulation when neutrophils were stimulated with phorbol myristate acetate or N-formylmethionyl-leucyl-phenylalanine. Cycloheximide did not affect the release of HGF. Moreover, HGF messenger RNA and protein expression was found in bone marrow myeloid cells, suggesting that HGF synthesis likely occurs during PMN maturation. In mature circulating PMNs, intracellular HGF was in the pro-HGF form, whereas the HGF secreted by degranulation was the mature form. Furthermore, PMNs pretreated with diisopropyl fluorophosphate only released the pro-HGF form, suggesting that PMN-derived serine protease(s) are involved in the proteolytic process. We also obtained evidence that secreted mature HGF binds PMN-derived glycosaminoglycans (probably heparan sulfate). These findings suggest that PMNs infiltrating damaged tissues may modulate local wound healing and repair through the production of HGF, a major mediator of tissue regeneration.
Randomized study of valacyclovir as prophylaxis against cytomegalovirus reactivation in recipients of allogeneic bone marrow transplants
Authors: Ljungman Per , de la Camara Rafael , Milpied Noel , Volin Liisa , Russell Charlotte A. , Crisp Adam , Webster Alison ,   the Valacyclovir International Bone Marrow Transplant Study Group
Published: October 2002

Oral valacyclovir for cytomegalovirus (CMV) prophylaxis in bone marrow transplantation (BMT) was investigated in a randomized, double-blind, acyclovir-controlled, multicenter clinical trial in recipients of allogeneic BMT who were CMV seropositive (or donor positive) before transplantation and were aged 13 years or older. Patients were randomized before BMT. All initially received intravenous acyclovir (500 mg/m2) 3 times daily until day 28 after transplantation or after discharge, then oral valacyclovir (2 g) or acyclovir (800 mg) 4 times daily until week 18 after transplantation. Evidence of CMV infection, CMV disease, and death were documented for 22 weeks. Primary end points were time to CMV infection (detection of CMV in blood, broncho-alveolar lavage) or CMV disease and survival. Preemptive CMV therapy was permitted. Seven hundred twenty-seven patients were evaluable for efficacy. After the administration of intravenous acyclovir, valacyclovir was significantly more effective than oral acyclovir in reducing the incidence of CMV infection. CMV infection or disease developed in 102 (28%) valacyclovir patients, compared with 143 (40%) acyclovir patients (HR, 0.59; 95% CI, 0.46-0.76; P < .0001). Survival did not differ between treatments (76% and 75% in the valacyclovir and acyclovir groups, respectively). The safety of oral valacyclovir was similar to that of high-dose oral acyclovir. Valacyclovir was more effective than acyclovir in preventing CMV reactivation in BMT recipients and showed a similar safety profile. CMV disease incidence was low, and no differences were observed between oral valacyclovir and acyclovir. Survival was similar in each group. Valacyclovir prophylaxis provides a clinically valuable intervention but must be part of an overall strategy for CMV prevention in BMT.
High-dose therapy and autologous blood stem cell transplantation in POEMS syndrome
Authors: Jaccard Arnaud , Royer Bruno , Bordessoule Dominique , Brouet Jean-Claude , Fermand Jean-Paul
Published: October 2002

We treated 5 patients with polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome and multifocal bone lesions or diffuse bone marrow plasmacytic infiltration with high-dose therapy (HDT) and autologous blood stem cell transplantation. In all cases, the treatment produced remission of plasma cell proliferation associated with marked improvement in the patients' performance status, neurologic symptoms, and other manifestations of the syndrome. HDT with stem cell support should be investigated further as a therapeutic option in patients with POEMS syndrome and disseminated plasma cell dyscrasia.
Functional amelioration of murine galactosialidosis by genetically modified bone marrow hematopoietic progenitor cells
Authors: Leimig Thasia , Mann Linda , Martin Maria del Pilar , Bonten Erik , Persons Derek , Knowles James , Allay James A. , Cunningham John , Nienhuis Arthur W. , Smeyne Richard , d'Azzo Alessandra
Published: October 2002

Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disorder galactosialidosis (GS). PPCA−/− mice display a disease course similar to that of severe human GS, resulting in nephropathy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overexpressing the corrective enzyme in either erythroid cells or monocytes/macrophages has proven effective for the improvement of the phenotype, and encouraged the use of genetically modified BM cells for ex vivo gene therapy of GS. Here, we established stable donor hematopoiesis in PPCA−/− mice that received hematopoietic progenitors transduced with a murine stem cell virus (MSCV)–based, bicistronic retroviral vector overexpressing PPCA and the green fluorescent protein (GFP) marker. We observed complete correction of the disease phenotype in the systemic organs up to 10 months after transplantation. PPCA+ BM-derived cells were detected in all tissues, with the highest expression in liver, spleen, BM, thymus, and lung. In addition, a lysosomal immunostaining was seen in nonhematopoietic cells, indicating efficient uptake of the corrective protein by these cells and cross-correction. Expression in the brain occurred throughout the parenchyma but was mainly localized on perivascular areas. However, PPCA expression in the central nervous system was apparently sufficient to delay the onset of Purkinje cell degeneration and to correct the ataxia. The long-term expression and internalization of the PPCA by cells of systemic organs and the clear improvement of the neurologic phenotype support the use of this approach for the treatment of GS in humans.
Constitutive and functional association of the platelet collagen receptor glycoprotein VI–Fc receptor γ-chain complex with membrane rafts
Authors: Ezumi Yasuharu , Kodama Kumi , Uchiyama Takashi , Takayama Hiroshi
Published: October 2002

The platelet collagen receptor glycoprotein (GP) VI–Fc receptor γ-chain (FcRγ) complex transduces signals in an immunoreceptorlike manner. We examined a role for the Triton X-100–insoluble membrane rafts in GPVI–FcRγ complex signaling. Methyl-β-cyclodextrin (MβCD)-induced disruption of the membrane rafts inhibited not only platelet aggregation and secretion but also tyrosine phosphorylation of signaling molecules on stimulation through the GPVI–FcRγ complex. The GPVI–FcRγ complex was constitutively associated with membrane rafts wherein the Src family kinases and LAT were also present. Their association was not affected by the complex engagement but was highly sensitive to MβCD treatment. Thus, we provide the first evidence that the GPVI–FcRγ complex is constitutively and functionally associated with membrane rafts.
Tumor lysate–specific cytotoxic T lymphocytes in multiple myeloma: promising effector cells for immunotherapy
Authors: Wen Yue-Jin , Min Rui , Tricot Guido , Barlogie Bart , Yi Qing
Published: October 2002

The idiotype protein, secreted by myeloma plasma cells, is a tumor-specific but weak antigen. Idiotype-based immunotherapy has been explored in myeloma patients with disappointing results. It is conceivable that myeloma cells contain a multitude of tumor antigens that can more effectively stimulate antitumor T cells. To explore the possibility of using whole myeloma cells as a source of tumor antigens for immunotherapy, the current study was undertaken to generate and examine the function of myeloma-specific cytotoxic T lymphocytes (CTLs) by using dendritic cells (DCs) pulsed with myeloma cell lysates as stimulating cells. After repeated stimulation, specific CTL lines, containing CD4+ and CD8+ T cells, were generated from myeloma patients. Our results show that these T cells not only recognized and lysed autologous myeloma protein–pulsed DCs, they also killed autologous primary myeloma cells. Occasionally, CTLs responded to autologous idiotype–pulsed DCs and to allogeneic primary myeloma cells. No cytolytic activity, however, was detected against autologous lymphocytes including B cells, suggesting that the T cells acted specifically against myeloma cells. Cytotoxicity against target cells was major histocompatibility complex class 1 and, to a lesser extent, class 2 restricted and was dependent mainly on the perforin-mediated pathway. CTLs secreted predominantly interferon-γ and tumor necrosis factor-α on antigenic stimulation, indicating a type 1 T-cell subset. These findings represent the first demonstration that tumor cell lysate–primed CTLs kill only myeloma cells, not autologous lymphocytes. This provides a rationale for myeloma cell–based immunotherapy in multiple myeloma.
Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells
Authors: Gray Christian P. , Arosio Paolo , Hersey Peter
Published: October 2002

Heavy chain ferritin (H-ferritin) is a component of the iron-binding protein, ferritin. We have previously shown that H-ferritin inhibits anti-CD3–stimulated lymphocyte proliferation and that this was due to increased production of interleukin-10 (IL-10). In the present study we have shown that induction of IL-10 production was due to effects of H-ferritin on adherent antigen-presenting cells (APCs) in blood and monocyte-derived dendritic cells (MoDCs). IL-10 was produced by a subpopulation of CD4 T cells, which expressed the CD25 component of the IL-2 receptor and the CTLA-4 receptor characteristic of regulatory T cells. The changes induced in MoDCs were compared with those induced by CD40L and their significance tested by inhibition with monoclonal antibodies. These studies indicated that H-ferritin induced relatively greater expression of CD86 and B7-H1 on MoDCs and that monoclonal antibodies against their receptors, CTLA-4 and programmed death receptor-1 (PD-1), inhibited IL-10 production from the regulatory T cells. H-ferritin did not appear to induce direct production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or interferon-γ from the DCs. These results are consistent with the thesis that H-ferritin induces B7-H1 and CD86 (B7-2) on APCs, which in turn induce IL-10 production from regulatory T cells. This is possibly one mechanism by which melanoma cells may induce changes in APCs in the vicinity of the tumor and result in suppression of immune responses by induction of regulatory T cells.